Lipid-based drug delivery systems against parasitic infections

ABSTRACT

The present invention is directed to a lipid-based delivery system for administration of an active drug substance selected from lysolipid derivatives which are particularly useful in the treatment or detection of parasitic infections, especially parasitic infections which causes an elevated PLA 2  level in the infected mammal. Preferred parasitic infection are infections wherein the life cycle of the parasite involves the liver and/or spleen of the infected organism.

This application is a Divisional of co-pending U.S. application Ser. No.10/239,527 filed on Jan. 23, 2003, for which priority is claimed under35 U.S.C. §120; and which is a U.S. National stage of internationalApplication No. PCT/DK01/00268, filed on Apr. 11, 2001; which claimspriority to foreign Application PA 2000 00616 filed in Denmark on Apr.12, 2000 under 35 U.S.C. §119; the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to lipid-based pharmaceutical compositions for usein the treatment or detection of parasitic infections.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 5,827,836 discloses retinoyl substitutedglycerophophoethanolamines. It is stated that the compounds and saltsthereof exhibit antitumor, anti-psoriatic and anti-inflammatoryactivities. A possible class of compounds has a fatty ether substituentin the 1-position, a retinoid ester (retinoyl) substituent in the2-position and a phosphoethanolamine substituent in the 3-position. Itis mentioned that some of the compounds can be presented in a liposomeformulation.

U.S. Pat. No. 4,372,949 discloses a carcinostatic and immunostimulatingagent containing a lysophospholipid and a phospholipid. Examples ofcompounds are 3-phosphorylcholine having a C₅₋₂₂-acyloxy or C₅₋₂₂-alkoxysubstituent in the 1-position, and a hydrogen, hydroxy, C₁₋₅-acyloxy orC₁₋₅-alkoxy substituent in the 2-position. It is mentioned that theagents can be dispersed in the form of micelles or lipid vesicles.

U.S. Pat. No. 5,484,911 discloses nucleoside 5′-diphosphate conjugatesof ether lipids which exhibit antiviral activity. The compounds may havea fatty ether/thioether substituent in the sn-1-position and a fattyacid ester substituent in the sn-2-position. The compounds are designedso as to penetrate the cell membrane whereafter the nucleoside drug isliberated by cleavage by intracellular phosphatases. It is furthermoresuggested that the also liberated ether lipids may be subsequentlycleaved by intracelluar phospholipase A₂. It is suggested that theconjugates can be presented in the form of micelles which more easilycan be taken up by macrophages.

U.S. Pat. No. 4,622,392 discloses cytotoxic compounds of thenucleotide-lipid conjugate type.

ES 2 034 884 discloses 2-aza-phospholipider as PLA₂ inhibitors.Similarly, de Haas et al (Biochem. Biophys. Acta, Lipid and LipidsMetabolism, 1167 (1993) No. 3, pp 281-288, discloses inhibition ofpancreatic PLA₂ by (R)-2-acylamino phospholipid analogues.

Hoffman et al., Blood, Vol. 63, No. 3 (March), 1984, pp 545-552,discloses the cytotoxicity of PAF and related alkyl-phospholipidanalogues in human Leukemia cells.

WO 94/09014 discloses phosphoric acid esters as PLA₂ inhibitors. A groupof the inhibitors are 1-O-phospho-2-O—(C₂₋₂₁-acyl)-(C₁₂₋₂₄-alkanes).

Xia and Hui discloses the chemical synthesis of a series of etherphospholipids from D-mannitol and their properties as tumor-cytotoxicagents.

U.S. Pat. No. 5,985,854, U.S. Pat. No. 6,077,837, U.S. Pat. No.6,136,796 and U.S. Pat. No. 6,166,089 describe prodrugs with enhancedpenetration into cells, which are particular useful for treating acondition or disease in a human related to supranormal intracellularenzyme activity. The prodrugs may be sn-2-esters of lysophospholipids.Such drugs are designed so as to be cleaved by intracelluarphospholipase A₂.

Vadas et al. (Infection and Immunity, 60 (1992) 3928-3931 and Am. J.Trop. Hyg. 49 (1993) 455-459) describe the induction of circulating PLA₂expression in humans with malaria infections caused by Plasmodiumfalciparum.

Lux et al. (Biochem. Parasitology 111 (2000) 1-14) describe theanti-leishmania action of certain ether-lipid analogues which isbelieved to be caused by the interference with important biosyntheses ofthe parasite.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to drug delivery systems which areparticularly useful in the treatment of parasitic infections especiallyparasitic infections wherein the life cycle of the parasite involves theliver and/or spleen of the infected organism. The liposomes of thepresent invention are only degraded to a very low extend by enzymespresent in the blood stream, whereas said liposomes are up-concentratedand degraded in the liver and/or spleen by the macrophages present inthese organs. This degradation of the liposomes releases constituentswhich are designed to be toxic for various types of parasites. In oneembodiment of the present invention the lysolipids released from theliposome are toxic to the parasites, in another embodimentanti-parasitic drugs are encapsulated in the liposomes. In a furtherembodiment a combination of liposomes made from lipids which are toxicto parasites in which anti-parasitic drugs are encapsulated.Accordingly, a highly efficient and indirectly specific release ofsubstances toxic to parasites is achieved in the case of parasiticinfection of tissues characterised by the presence of macrophages, suchas the liver and/or the spleen.

Yet another embodiment of the present invention is the diagnostic use ofthe presently described drug delivery systems, wherein the drug deliverysystem carries a label which, by the action described above, isspecifically directed towards the tissue or organs being infected by theparasite.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Heat capacity curves obtained using differential scanningcalorimetry. (a) Multilamellar, MLV (the upper curve) and unilamellar,LUV (the bottom curve) liposomes made of 1 mM1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (1-O-DPPC). (b)MLV (the upper curve) and LUV (the bottom curve) liposomes made ofdipalmitoylphosphatidylcholine (DPPC).

FIG. 2. Characteristic reaction time profile at 41° C. for phospholipaseA₂, PLA₂, (A. piscivorus piscivorus) hydrolysis of unilamellar liposomescomposed of 1-O-DPPC. The PLA₂ hydrolysis reaction is monitored byintrinsic fluorescence (solid line) from the enzyme and 90° static lightscattering (dashed lines) from the lipid suspension. After adding PLA₂,at 800 sec to the equilibrated liposome suspension a characteristiclag-time follows before a sudden increase in the catalytic activitytakes place. Samples for HPLC were taken before adding the enzyme and 20minutes after the observed lag time.

FIG. 3. HPLC chromatograms illustrating the effect of phospholipase A₂hydrolysis of liposomes composed of 1-O-DPPC. The chromatograms show theamount of 1-O-DPPC (100%, solid line) before phospholipase A₂ (A.piscivorus piscivorus) was added to the liposome suspension and theamount of 1-O-DPPC (21%, dashed line) after the lag-burst.

FIG. 4. PLA₂-controlled release of the fluorescent model drug calceinfrom liposomes composed of 25 μM1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (1-O-DPPC)suspended in a 10 mM HEPES-buffer (pH=7.5), as a function of time. 25 nMphospholipase A₂ (A. piscivorus piscivorus) was added at time 900 sec,the temperature was 37° C. The percentage of calcein released isdetermined as % Release=100 (I_(F(t))−I_(B))/(I_(T)−I_(B)), whereI_(F(t)) is the measured fluorescence at time t after addition of theenzyme, I_(B) is the background fluorescence, and I_(T) is the totalfluorescence measured after addition of Triton X-100 which leads tocomplete release of calcein by breaking up the liposomes.

FIG. 5. PLA₂-controlled release of the fluorescent model drug calceinacross the target membrane of non-hydrolysable membranes (see FIG. 11b), as a function of time for liposomes composed of 25 μM1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (1-O-DPPC)suspended in a 10 mM HEPES-buffer (pH=7.5). 25 nM phospholipase A₂ wasadded at time 0 sec and the temperature was 37° C. The percentage ofcalcein released is determined as described in FIG. 4.

FIG. 6. Hemolysis profile of normal red blood cells in the presence ofliposomes composed of 100% 1-O-DPPC (squares); 95% 1-O-DPPC and 5%negatively charged1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350] (1-O-DPPE-PEG350) (triangles) and1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH₃)(diamonds). The concentrations that yield 5% hemolysis (H₅) were wellabove 2 mM for liposomes composed of 100% 1-O-DPPC, and for liposomescomposed of 90% 1-O-DPPC with 5% DPPE-PEG350. Hemolysis assay wasperformed as described by Perkins et al., 1997, Biochimica et BiophysicaActa 1327, 61-68. Briefly, each sample was serially diluted withphosphate buffered saline (PBS), and 0.5 ml of each dilute suspensionwas mixed with 0.5 ml washed human red blood cells (RBC) [4% in PBS(v/v)]. Sample and standard were placed in a 37° C. incubator andagitated for 20 hours. Tubes were centrifuged at low speed (2000×G) for10 minutes and 200 μl of the supernatant was quantitated by absorbanceat 550 nm. 100 percent hemolysis was defined as the maximum amount ofhemolysis obtained from the detergent Triton X-100. The hemolysisprofile in FIG. 6 shows a low hemolysis value (below 5% percent) for 2mM 1-O-DPPC-liposomes and for 1-O-DPPC with 5% 1-O-DPPE-PEG350,liposomes.

FIG. 7. Characteristic reaction time profiles at 41° C. for PLA₂ (A.piscivorus piscivorus) hydrolysis of unilamellar liposomes incorporatedwith 0 and 5% 1-O-DPPE-PEG350 lipopolymers. The PLA₂ hydrolysis reactionis monitored by intrinsic fluorescence (solid line) from the enzyme and90° static light scattering (dashed lines) from the suspension. Afteradding PLA₂ to the equilibrated liposome suspension a characteristiclag-time follows before a sudden increase in the catalytic activitytakes place.

FIG. 8. PLA₂-controlled release of the fluorescent model drug calceinacross the target membrane of non-hydrolysable D-O-SPC membranes as afunction of time for micelles composed of 25 μM 1-O-DPPE-PEG350 (dottedline), DSPE-PEG750 (dashed line) suspended in a 10 mM HEPES-buffer(pH=7.5). Phospholipase A₂ (25 nM) was added at time 1200 sec and thetemperature was 41° C. The percentage of calcein released is determinedas described in FIG. 4. PLA₂ catalysed hydrolysis of 1-O-DPPE-PEG350induced the fastest and highest release.

FIG. 9. HPLC chromatograms illustrating the effect of phospholipase A₂hydrolysis of micelles composed DSPE-PEG750 (0.150 mM). Thechromatograms show the amount of stearic acid generated before (solidline) phospholipase A₂ (A. piscivorus piscivorus) was added to themicelle suspension and the amount (dashed line) of DSPE-PEG750 after thelag-burst. The dotted line show pure stearic acid (0.4 mM). Thepercentage hydrolysis was calculated on basis of the integrated area ofthe stearic acid standard (115850 units) and the integrated area of thesample (45630 units). The concentration of the stearic acid in thesample was calculated to (45630/115850×0.4 mM) 0.157 mM, which meansthat 100% of the DSPE-PEG750 was hydrolysed to lyso-SPE-PEG750 andstearic acid.

FIG. 10. Describes the principle of liposomal drug targeting, releaseand absorption by extracellular enzymes.

-   -   (I) Reticuloendothelial system, e.g. macrophages in liver and        spleen infected with parasites (i.e. Leishmania)    -   (II) Prodrug carrier liposome    -   (III) Target parasitic cell and cell membrane    -   (IV) Prodrug (i.e. monoether-lipid), proenhancer (lipid),        proactivator (lipid)    -   (V) Drugs (i.e. ether-lysolipid and fatty acid derivatives)        enhancers (lysolipid+fatty acid) PLA₂ activators        (lysolipid+fatty acid)

FIG. 11 Schematic illustration of a liposomal drug-targeting principleinvolving accumulation of the liposomal drug carriers the RES in porousdiseased tissue and subsequent release of drug and transport across thetarget membrane via extracellular PLA₂ activity.

-   -   (I) Prodrug carrier liposome    -   (II) Non-degradable target liposomal membrane    -   (III) Non-hydrolysable ether-lipids    -   (IV) Proenhancer (lipid), prodrug (i.e. monoether-lipid),        proactivator (lipid)    -   (V) Enhancers (lysolipid+fatty acid), drugs (i.e.        ether-lysolipid and fatty acid derivatives), PLA₂ activators        (lysolipid+fatty acid)

FIG. 12 (a) PLA₂-controlled release of the fluorescent model drugcalcein across the target membrane as a function of time for differentcompositions of the carrier liposomes. The temperature is 37° C. Incomparison with bare DPPC carriers, the rate of release of the modeldrug is dramatically enhanced for the carriers incorporated with 2.5 mol% of the negatively charged DPPE-PEG2000. A further augmentation of therate of release is obtained if the carrier also contains a short-chainphospholipid, didecanoylphosphatidylcholine (DCPC), which acts as alocal activator for the enzyme. The percentage of calcein released isdetermined as % Release=100 (I_(F(t))−I_(B))/(I_(T)−I_(B)), whereI_(F(t)) is the measured fluorescence at time t after addition of theenzyme, I_(B) is the background fluorescence, and I_(T) is the totalfluorescence measured after addition of Triton X-100 which leads tocomplete release of calcein by breaking up the target liposomes. (b)PLA₂-controlled release of the fluorescent model drug calcein across thetarget membrane as a function of time for different temperatures. As thetemperature is raised, the rate of release is enhanced due to increasedactivity of the enzyme induced by structural changes in the lipidbilayer substrate of the carrier liposome. In the present assay amaximum release of about 70% is achieved in all cases. The insert showsthe time of 50% calcein release, t_(50%), as a function of temperature.The concentration of the target and carrier liposomes are 25 μM, andPLA₂ is added in a 25 nM concentration in a HEPES buffer with pH=7.5.

FIG. 13. Total release after 20 min of the fluorescent model drugcalcein across the target membrane as a function of adding increasingamounts of lyso-palmitoyl phospholipid and palmitic acid, separately,and in a 1:1 mixture. The concentration of the target membranes is 25 μMin a HEPES buffer with pH=7.5 at a temperature of 39° C.

FIG. 14. PLA₂-controlled release of the fluorescent model drug calceinfrom liposomes composed of 25 μM 90 mol % 1-O-DPPC and 10 mol % of thenegatively charged 1-O-DPPE-PEG350 suspended in a 10 mM HEPES-buffer(pH=7.5), as a function of time. 50 nM (straight line), 1 nM (solidline) and 0.02 nM (dotted line) phospholipase A₂ (A. piscivoruspiscivorus) was added at time 300 sec, the temperature was 35.5° C. Thepercentage of calcein released is determined as describe in FIG. 4.

FIG. 15. Emission spectrum of 1 mol % bis-py-DPC incorporated innegatively charged liposomes (0.100 mM) before (solid line) and after(dashed line) adding 100 nM PLA₂ (Agkistrodon piscivorus piscivorus) toa liposome suspension equilibrated at 41° C.

FIG. 16. Characteristic reaction time profile at 41° C. for ratphospholipase catalysed hydrolysis of negatively charged liposomes. Thecatalytic reaction was initiated by adding cell-free peritoneal fluid to2.5 ml of the thermostated liposome suspension equilibrated for 60 secprior to addition of peritoneal fluid. The hydrolysis reaction ismonitored by monomer fluorescence (solid line) and eximer fluorescence(dashed line) from bis-py-DPC. After adding undiluted peritoneal fluid,at 60 sec, to the equilibrated liposome suspension a sudden increase inmonomer fluorescence, and a simultaneously decrease in eximerfluorescence is observed as the bis-py-DPC substrate is hydrolysed. Theinsert shows the reaction time profile of the first 120 sec.

DETAILED DESCRIPTION OF THE INVENTION

One of the important features of the present invention is therealisation of the rapid uptake of liposomes in vivo by cells of themononuclear phagocytic system (MPS). The MPS comprises the macrophages,one of the most important components of the immune system especially inclearance of foreign particles, including liposomes. The macrophagesresides in various organs and tissues, e.g. in connective tissue (ashistiocytes), in the liver (Kupffer cells) and as free and fixedmacrophages in the spleen, bone marrow and lymph nodes.

When liposomes in general are administered intravenously they aretypically removed from the circulation by the macrophages of the liver,spleen and bone marrow. This removal consists of a first opsonization byblood proteins followed by macrophage uptake of these marked liposomes.The immune opsonins are comprised by immunoglobulins and complementproteins, and although each opsonin has its own interaction and confersdifferent fates, it seems that liver sequestration is complementmediated and the spleen removes the opsonized liposomes in general. Theenhanced clearance of the liposomes of the present invention obtained bythe opsonization can be characterised as passive targeting.

As the MPS uptake of liposomes results in the accumulation of theliposomes in tissues like the liver, the spleen and the bone marrow itenables a targeting directed against parasitic infections of thesetissues. The liposomes of the present invention comprises constituentswhich can act as a label for detecting parasite infected tissue/organsor can act as anti-parasitic drugs when released from the liposomes tothe tissues of a mammal suffering from a parasitic infection.

One embodiment of the present invention is thus a method for thetreatment of parasitic infections which is characterised by an increasedlevel of PLA₂ in a mammal, preferably a human, by administering to themammal an efficient amount of a lipid-based delivery system foradministration of an active drug substance selected from lysolipidderivatives, wherein the active drug substance is present in thelipid-based system in the form of a prodrug, said prodrug being a lipidderivative having (a) an aliphatic group of a length of at least 7carbon atoms and an organic radical having at least 7 carbon atoms, and(b) a hydrophilic moiety, said prodrug furthermore being a substrate forextracellular phospholipase A2 to the extent that the organic radicalcan be hydrolytically cleaved off, whereas the aliphatic group remainssubstantially unaffected, whereby the active drug substance is liberatedin the form of a lysolipid derivative which is not a substrate forlysophospholipase.

A further embodiment is a method for the treatment of parasiticinfections which is characterised by an increased level of PLA₂ in amammal, preferably a human, by administering to the mammal an efficientamount of the lipid based delivery system described above foradministration of an second substance, wherein the second substance is aanti-parasitic drug incorporated in said system.

In one specific embodiment of the present invention the increased levelof PLA₂ is localized to a specific tissue and/or organ of the mammal,said tissue and/or organ being infected by the parasite. Especially asituation wherein the parasitic infection involves the liver and/or thespleen and/or the bone marrow of the mammal is comprised by the presentinvention.

The treatment is preferably performed by systemically administration ofthe lipid based delivery system of the present invention, even morepreferably administered parenterally by injection such as intravenousinjection.

Yet another embodiment of the present invention is a method fordiagnosing a parasitic infection which is characterised by an increasedlevel of PLA₂ of the infected tissue by administering to the mammal anefficient amount of the lipid based delivery system described above foradministration of an second substance, wherein the second substance is alabel incorporated in said system. By administering such a construct toa patient suspected of having a parasitic infection it is possible todetermine if the patient is infected and in the case of a parasiticinfection the localized areas of the patients body harbouring theparasite is identified. The localized deliverance of the diagnosticagent—the label—enables the use of diagnostic imaging tools such aspositron emission tomography (PET), X-ray, gamma-scintigraphy, magneticresonance (MR) imaging, computed tomography (CT) imaging andultrasonography.

The labels applicable for the medical imaging are selected from thegroup consisting of diagnostic radionuclides, such as ¹¹¹In, ^(99m)Tc,⁶⁷Ga, ¹¹C; paramagnetic ions, such as Gd and Mn, and iron oxide; gas,such as air, argon, nitrogen; Iodine; bromine and barium.

Target Parasitic Organisms

The liposomes of the present invention are able to deliver variousether-lipid analogues to tissues harbouring parasites due to increasedlevels of PLA₂ levels of said tissue. The ether-lipid analogues havebeen shown to result in perturbation of key enzymes involved in e.g.alkyl-phospholipid biosynthesis of parasites such as Leishmania andTrypanosomas (Lux et al. Biochem. Parasitology 111 (2000) 1-14) and aretherefore toxic to the parasites if administered in sufficient amount.

As the liver, spleen and bone marrow are organs/tissues wherein highconcentrations of macrophages can be found, targeting parasitesinhabiting these organs is preferred because of resulting accumulationof liposomes of the present invention, but other tissues characterizedby having increased PLA₂ levels during parasitic infection may also beof interest.

Furthermore, parasitic infections which are characterised by highlyelevated levels of circulating PLA₂, such as malaria-causing parasites,e.g. caused by Plasmodium falciparum, are also targets for treatmentwith liposomes of the present invention. This characteristic of elevatedlevels of circulating PLA₂ of malaria infections caused by the parasitePlasmodium falciparum has been described by e.g. Vadas et al. (Infectionand Immunity, 60 (1992) 3928-3931 and Am. J. Trop. Hyg. 49 (1993)455-459).

Examples of parasite infections resulting in increased PLA₂ level of theparasite harbouring tissue are given below.

Leishmania

Members of the genus Leishmania has the potential to infect variousvertebrate species, including humans, dogs, and rodents and the varioustypes of leishmaniasis are confined primarily, but not exclusively, toCentral and South America, central Africa, and parts of southern andcentral Asia.

The life cycle of members of the genus involve a vertebrate host e.g.the human and a vector that transmits the parasite between vertebratehosts. In the case of Leishmania the vector is various species ofPhlebotomus sand flies.

The characteristic morphological form taken by the Leishmania paracitein the vector is the promastigote, and in this stage it reproducesasexually in the vector's gut. Upon biting the vertebrate host,promastigotes from the vector are injected into the vertebrate host.After the entrance into the vertebrate host the promastigotes changeinto a form called amastigote. The amastigote reproduces in the host'scells, and when the vertebrate host cell eventually dies, theamastigotes are released and will potentially infect other cells. Thesymptoms and pathology associated with leishmaniasis result from theamastigotes killing the host's cells.

Leishmania is the cause of several different conditions depending on thesite of infection of the vertebrate host. In some diseases theamastigotes do not spread beyond the site of the vector's bite whichthen results in a “cutaneous leishmaniasis” also known as oriental sore,Jericho boil, Aleppo boil, or Dehli boil. These conditions often healspontaneously. In other instances the amastigotes may spread to thevisceral organs, i.e. the liver and the spleen, which results in“visceral leishmaniasis” also known as kala-azar or Dum-Dum fever.Furthermore, the amastigotes may spread to the mucous membranes of themouth and nose, resulting in the condition of “mucocutaneousleishmaniasis” also known as espundia or uta. Left untreated, theselatter diseases result in high rates of mortality.

Non-limiting examples of Leishmania species are Leishmania majorFriedlin, Leishmania (viannia) gr., Leishmania mexicana, Leishmaniatropica, Leishmania donovani (infantum), Leishmania aethiopica,Leishmania amazonensis, Leishmania enrieftii, Leishmania chagasi andLeishmania pifanoi.

Traditional anti-leishmanial therapy agents are compounds like thepentavalent antimony compounds sodium stibogluconate (Pentosram™) andmeglumine antimonate (Glucantime™), other drugs are amphotericin,metronidazole, allopurinol and pentamidine, furthermore paromomycin andinterferon gamma.

Both for malaria treatment and Leishmania treatment a new drug,Licochocone A, which was originally isolated from the roots of Chineselicorice, has been proposed.

Trypanosoma

Three main species of trypanosomes causes disease in humans, these areTrypanosoma gambiense and Trypanosoma rhodesiense, which both causesleeping sickness in Africa, and Trypanosoma cruzi, which causesChargas' disease in South America.

Both types of disease are characterised by bouts of parasitaemia andfever. The damage to the organs is caused toxins released by theparasites.

The most common vector for transmitting the trypanosomes causingsleeping sickness is the tsetse fly Glossina sp. The species that causehuman African trypanosomiasis (sleeping sickness) also infect wildanimals and can be transmitted from these animals to humans (zoonoticinfections).

In humans, Trypanosoma cruzi is found as both an intracellular form, theamastigote, and as a trypomastigote form in the blood. The vector forChagas' disease is a “true bug” (Hemiptera) such a Triatoma, Rhodnius,or Panstongylus which ingests amastigotes or trypomastigotes when itfeeds. In the vector the parasite reproduces asexually and metacyclictrypomastigotes are found in the vector's hindgut. The vector defecateson the host's skin at the same time that it feeds, and the metacyclictrypomastigotes enter the host's body, most often by being “rubbed in”to the vector's bite or the mucous membranes of the eye, nose, or mouth.In the human host, Chagas' disease affects primarily the nervous systemand heart but also sometimes the liver, spleen, bone and intestine.Chronic infections result in various neurological disorders, includingdementia, megacolon, and megaesophagus, and damage to the heart muscle.Left untreated, Chagas' disease is often fatal.

The main traditional drugs used for treatment of sleeping sickness aresuramin and pentamidine. Traditional drugs for Chargas' disease areprimaquine, puromycin and nitrofurantoin derivatives, but none of thesedrugs have proven really effective in the treatment for this condition.

Non-limiting examples of Trypanosoma species are Trypanosoma cruzi(causing Chargas' disease), Trypanosoma brucei, Trypanosoma equiperdumand Trypanosoma evansi.

Plasmodium—Malaria

Malaria is one of the major killer diseases of the world causing anestimated one-two million deaths annually. Malaria is caused by variousspecies of plasmodia. The female anopheline mosquito injectssporozoites, which in the liver can develop into merozoites. Themerozoites infect red blood cells and in the red blood cells themerozoites grow and eventually cause the cell to rupture and releasemore merozoites most of which infect other red blood cells and repeatthe cycle. Some of the merozoites evolve into gametocytes, which infectthe female anopheline mosquito.

The rupture of the blood cells and the release of merozoites cause thefever associated with malaria. The frequency of the fever attacksdepends on the species of Plasmodium. Plasmodium falciparum, Plasmodiumvivax and Plasmodium ovale causes red blood cell rupture after 48 hrs.and the malaria patient therefor experiences violent fever attacks everythird day. Plasmodium malariae causes red blood cell rupture every 72hrs.

Entamoeba histolytica

This protozoan parasite infects the lower bowel and frequently causesamebic dysentery. If untreated can lead to death. This amoebae is notrestricted to the large intestine but can spread to other soft organs,particularly the liver, where it can produce large abscesses over thecourse of a few years. People become infected by ingesting the amebiccyst in contaminated food or water.

The Oriental Liver Fluke:

The life cycle of the oriental liver fluke Chlornorchis sinensis startswith a miracidium infecting a snail. In the snail the miracidiumdevelops into a cercariae. The cercariae leaves the snail and penetratethe skin of a fish. In the fish the cercariae encrysts itself in themussel tissue and when the fish raw or undercooked fish is eaten theimmature worm is released and it migrates to the liver where it maturescausing damage to the liver.

Lipid Derivatives

The lipid-based delivery systems (liposomes or micelles) relies on lipidderivatives having (a) an aliphatic group of a length of at least 7carbon atoms and an organic radical having at least 7 carbon atoms, and(b) a hydrophilic moiety, said prodrug furthermore being a substrate forextracellular phospholipase A2 to the extent that the organic radicalcan be hydrolytically cleaved off, whereas the aliphatic group remainssubstantially unaffected, whereby the active drug substance is liberatedin the form of a lysolipid derivative which is not a substrate forlysophospholipase.

Although the terms “lipid” and “lysolipid” (in the context ofphospholipids) will be well-known terms for the person skilled in theart, it should be emphasised that, within the present description andclaims, the term “lipid” is intended to mean triesters of glycerol ofthe following formula:

wherein R^(A) and R^(B) are fatty acid moieties(C₉₋₃₀-alkyl/alkylene/alkyldiene/alkyltriene/-alkyltetraene-C(═O)—) andR^(C) is a phosphatidic acid (PO₂—OH) or a derivative of phosphatidicacid. Thus, the groups R^(A) and R^(B) are linked to the glycerolbackbone via ester bonds.

The term “lysolipid” is intended to mean a lipid where the R^(B) fattyacid group is absent (e.g. hydrolytically cleaved off), i.e. a glycerolderivative of the formula above where R^(B) is hydrogen, but where theother substituents are substantially unaffected. Conversion of a lipidto a lysolipid can take place under the action of an enzyme,specifically under the action of cellular as well as extracellular PLA₂.

The terms “lipid derivative” and “lysolipid derivative” are intended tocover possible derivatives of the above possible compounds within thegroups “lipid” and “lysolipid”, respectively. Examples of biologicallyactive lipid derivatives and lysolipid derivatives are given inHoulihan, et al., Med. Res. Rev., 15, 3, 157-223. Thus, as will beevident, the extension “derivative” should be understood in the broadestsense.

Within the present application, lipid derivatives and lysolipids shouldhowever fulfil certain functional criteria (see above) and/or structuralrequirements. It is particularly relevant to note that the suitablelipid derivatives are those which have (a) an aliphatic group of alength of at least 7, preferably at least 9, carbon atoms and an organicradical having at least 7 carbon atoms, and (b) a hydrophilic moiety. Itwill be evident that the aliphatic group and the organic radical willcorrespond to the two fatty acid moieties in a normal lipid and that thehydrophilic moiety will correspond to the phosphate part of a(phospho)lipid or a bioisoster thereof.

Thus, one element of the idea behind the present invention is to exploitthe increased level of extracellular PLA₂ activity in localised areas ofthe body of a mammal, in particular areas of parasitic infection, thelipid derivatives which can be utilised within the present inventionshould be substrates for extracellular PLA₂, i.e. the lipid derivativesshould be able to undergo hydrolytic, enzymatic cleavage of the organicradical corresponding to the fatty acid in the 2-position in a lipid.Extracellular PLA₂ is known to belong to the enzyme class (EC) 3.1.1.4.Thus by reference to (extracellular) PLA₂ should be understood allextracellular enzymes of this class, e.g. lipases, which can inducehydrolytic cleavage of the organic radical corresponding to the fattyacid in the 2-position in a lipid. One particular advantage of thelipid-based delivery system (as liposomes and micelles) is thatextracellular PLA₂ activity is significantly increased towards organisedsubstrates as compared to monomeric substrates.

In view of the requirement to hydrolysability by extracellular PLA₂, itis clear that the organic radical (e.g. aliphatic group) is preferablylinked via an ester functionality which can be cleaved by extracellularPLA₂, preferably so that the group which is cleaved off is a carboxylicacid.

Furthermore, an important feature is that the aliphatic group (the groupcorresponding to the fatty acid in the 1-position in a lipid) of thelipid derivative, i.e. the lysolipid derivative after cleavage byextracellular PLA₂, is substantially unaffected by the action ofextracellular PLA₂. By “substantially unaffected” is meant that theintegrity of the aliphatic group is preserved and that less than 1 mol%, preferably less than 0.1 mol %, of the aliphatic group (the aliphaticgroup in the 1-position) is cleaved under the action of extracellularPLA₂.

Also, the lysolipid derivative resulting from the hydrolytic cleavage ofthe organic radical should not in itself be a substrate forlysophospholipase. Lysophospholipase is known to belong to the enzymeclass (EC) 3.1.1.5. Thus by reference to lysophospholipase should beunderstood all enzymes of this class that catalyses the reactionlyso(phospho)lipid+water yielding phosphoglycerol+fatty acid. The term“not a substrate for lysophospholipase” is intended to mean thatlysophospholipase has an activity of less than 1% towards the substratecompared with the corresponding esterlipid, i.e. virtually not enzymaticactivity.

Suitable examples of such lysolipid derivatives are those which will notundergo hydrolytical cleavage under the action of lysophospholipases.Thus, the lysolipid derivatives are in particular not lysolipids andlysolipid derivatives which have an ester linkage in the 1-position ofthe lysolipid or the position of a lysolipid derivative whichcorresponding to the 1-position of a lysolipid.

One preferred class of lipid derivatives for incorporation in thelipid-based delivery systems can be represented by the followingformula:

wherein

X and Z independently are selected from O, CH₂, NH, NMe, S, S(O), andS(O)₂, preferably from O, NH, NMe and CH₂, in particular O;

Y is —OC(O)—, Y then being connected to R² via either the oxygen orcarbonyl carbon atom, preferably via the carbonyl carbon atom;

R¹ is an aliphatic group of the formula Y¹Y²;

R² is an organic radical having at least 7 carbon atoms, such as analiphatic group having a length of at least 7, preferably at least 9,carbon atoms, preferably a group of the formula Y¹Y²;

where Y¹ is—(CH₂)_(n1)—(CH═CH)_(n2)—(CH₂)_(n3)—(CH═CH)_(n4)—(CH₂)_(n5)—(CH═CH)_(n6)—(CH₂)_(n7)—(CH═CH)_(n8r)—(CH₂)_(n9),and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 to29; n1 is zero or an integer of from 1 to 29, n3 is zero or an integerof from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero oran integer of from 1 to 14, and n9 is zero or an integer of from 1 to11; and each of n2, n4, n6 and n8 is independently zero or 1; and Y² isCH₃ or CO₂H; where each Y¹—Y² independently may be substituted withhalogen or C₁₋₄-alkyl, but preferably Y¹—Y² is unsubstituted,

R³ is selected from phosphatidic acid (PO₂—OH), derivatives ofphosphatidic acid and bioisosters to phosphatic acid and derivativesthereof.

As mentioned above, preferred embodiments imply that Y is —OC(O)— whereY is connected to R² via the carboxyl atom. The most preferredembodiments imply that X and Z are O and that Y is —OC(O)— where Y isconnected to R² via the carboxyl atom. This means that the lipidderivative is a 1-monoether-2-monoester-phospholipid type compound.

Another preferred group of lipid derivatives is the one where the groupX is S.

In one embodiment, R¹ and R² are aliphatic groups of the formula Y¹Y²where Y² is CH₃ or CO₂H, but preferably CH₃, and where Y¹ is—(CH₂)_(n1)(CH═CH)_(n2)(CH₂)_(n3)(CH═CH)_(n4)—(CH₂)_(n5)(CH═CH)_(n6)(CH₂)_(n7)(CH═CH)_(n8)(CH₂)_(n9);the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 to 23;that is, the aliphatic group, Y¹Y², is from 10-24 carbon atoms inlength. n1 is equal to zero or is an integer of from 1 to 23; n3 isequal to zero or is an integer of from 1 to 20; n5 is equal to zero oris an integer of from 1 to 17; n7 is equal to zero or is an integer offrom 1 to 14; n9 is equal to zero or is an integer of from 1 to 11; andeach of n2, n4, n6 and 8 is independently equal to zero or 1.

In one embodiment, one or more of the aliphatic groups R¹/R² or the R³groups include a label, e.g. halogens (bromo, iodo) or barium atomswhich are particular suitable for computed tomography (CT) imaging, orare enriched with unstable isotopes, e.g. ¹¹C which is particularlyuseful for PET scanning purposes.

Although the aliphatic groups may be unsaturated and even substitutedwith halogens (flouro, chloro, bromo, iodo) and C₁₋₄-groups (i.e.yielding branched aliphatic groups), the aliphatic groups as R¹ and R²are in one embodiment preferably saturated as well as unbranched, thatis, they preferably have no double bonds between adjacent carbon atoms,each of n2, n4, n6 and n8 then being equal to zero. Accordingly, Y¹ ispreferably (CH₂)_(n1). More preferably (in this embodiment), R¹ and R²are each independently (CH₂)_(n1)CH₃, and most preferably, (CH₂)₁₇CH₃ or(CH₂)₁₅CH₃. In alternative embodiments, the groups can have one or moredouble bonds, that is, they can be unsaturated, and one or more of n2,n4, n6 and n8 can be equal to 1. For example, when the unsaturatedhydrocarbon has one double bond, n2 is equal to 1, n4, n6 and n8 areeach equal to zero and Y¹ is (CH₂)_(n1)CH═CH(CH₂)_(n3). n1 is equal tozero or is an integer of from 1 to 21, and n3 is also zero or is aninteger of from 1 to 20, at least one of n1 or n3 not being equal tozero.

In one particular embodiment, the lipid derivatives are those which aremonoether lipids where X and Z are O, R¹ and R² are independentlyselected from alkyl groups, (CH₂)_(n)CH₃, where n is 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, preferably14, 15 or 16, in particular 14; Y is —OC(O)—, Y then being connected toR² via the carbonyl carbon atom.

With respect to the hydrophilic moiety (often known as the “head group”)which corresponds to R³, it is believed that a wide variety of groupscorresponding to phosphatidic acid (PO₂—OH), derivatives of phosphatidicacid and bioisosters to phosphatic acid and derivatives thereof can beused. As will be evident, the crucial requirement to R³ is that thegroups should allow for enzymatic cleavage of the R² group (actuallyR²—C(═O) or R²—OH) by extracellular PLA₂. “Bioisosters to phosphatidicacid and derivatives thereof” indeed implies that such groups—asphosphatidic acid—should allow for enzymatic cleavage by extracellularPLA₂.

R³ is typically selected from phosphatidic acid (PO₂—OH),phosphatidylcholine (PO₂—O—CH₂CH₂N(CH₃)₃), phosphatidylethanolamine(PO₂—O—CH₂CH₂NH₂), N-methyl-phosphatidylethanolamine (PO₂—O—CH₂CH₂NCH₂),phosphatidylserine, phosphatidyl-inositol, and phosphatidylglycerol(PO₂—O—CH₂CHOHCH₂OH). Other possible derivatives of phosphatidic acidare those where dicarboxylic acids, such as glutaric, sebacic, succinicand tartaric acids, are coupled to the terminal nitrogen ofphosphatidylethanolamines, phosphatidylserine, phosphatidylinositol,etc.

One highly interesting aspect is the possibility of modifying thepharmaceutical effect of the lipid derivative by modifying the group R².It should be understood that R² should be an organic radical having atleast 7 carbon atoms) (such as an aliphatic group having a certainlength (at least 7, preferably 9, carbon atoms)), a high degree ofvariability is possible, e.g. R² need not necessarily to be a long chainresidue, but may represent more complex structures.

Generally, it is believed that R² may either be rather inert for theenvironment in which it can be liberated by extracellular PLA₂ or thatR² may play an active pharmaceutical role, typically as an auxiliaryanti-parasitic drug substance, such as allopurinol, amodiaquine,amphotericin, antifolates, artemether+benflumetol combination,artemisinin, derivatives, chloroproguanil, Chloroquine, combination ofatovaquone and proguanil HCL (salesname Malarone™), dapsone,Doxycycline, halofantrine, interferon gamma, Licochalcone A, Mefloquine,meglumine antimonate, metronidazole, nitrofurantoin derivatives,paromomycin, pentamidine, primaquine, primaquine, Proguanil,(Chloroquine plus proguanil), puromycin, pyrimethamine, pyronaridine,quinine and sodium stibogluconate or as an efficiency modifier for thelysolipid derivative and/or any other (second) substances present in theenvironment.

In one embodiment, one or more of the aliphatic groups R¹/R² or the R³groups include a label, e.g. halogens (bromo, iodo) or barium atomswhich are particular suitable for computed tomography (CT) imaging, orare enriched with unstable isotopes, e.g. ¹¹C which is particularlyuseful for PET scanning purposes.

In some embodiments, the group R² will be a long chain residue, e.g. afatty acid residue (the fatty acid will include a carbonyl from thegroup Y). This has been described in detail above. Interesting examplesof auxiliary drug substances as R² within this subgroups arepolyunsaturated acids, e.g. oleate, linoleic, linolenic, as well asderivatives of arachidonoyl (including the carbonyl from Y), e.g.prostaglandins such as prostaglandin E₁, as arachidonic acid derivativesare know regulators of hormone action including the action ofprostaglandins, thromboxanes, and leukotrines. Examples of efficiencymodifiers as R² are those which enhance the permeability of the targetcell membrane as well as enhances the activity of extracellular PLA₂ orthe active drug substance or any second drug substances. Examples hereofare short chain (C₈₋₁₂) fatty acids.

However, it is also envisaged that other groups might be useful as theorganic radical R², e.g. vitamin D derivatives, steroid derivatives,retinoic acid (including all-trans-retinoic acid, all-cis-retinoic acid,9-cis-retinoic acid, 13-cis-retinoic acid), cholecalciferol andtocopherol analogues, pharmacologically active carboxylic acids such asbranched-chain aliphatic carboxylic acids (e.g. valproic acid and thosedescribed in WO 99/02485), salicylic acids (e.g. acetylsalicylic acid),steroidal carboxylic acids (e.g. lysergic and isolysergic acids),monoheterocyclic carboxylic acids (e.g. nicotinic acid) andpolyheterocyclic carboxylic acids (e.g. penicillins and cephalosporins),diclofenac, indomethacin, ibuprofen, naproxen,6-methoxy-2-naphthylacetic acid.

It should be understood that the various examples of possible R² groupsare referred to by the name of a discrete species, rather than the nameof the radical. Furthermore, it should be understood that the possibleexamples may include the carbonyl group or oxy group of the bond viawhich the organic radical is linked to the lipid skeleton (correspondingto “Y” in the formula above). This will of course be appreciated by theperson skilled in the art.

Even though it has not specifically been indicated in the generalformula for the suitable examples of lipid derivatives to be used withinthe present invention, it should be understood that the glycol moiety ofthe lipid derivatives may be substituted, e.g. in order to modify thecleavage rate by extracellular PLA₂ or simply in order to modify theproperties of the liposomes comprising the lipid derivatives.

Lipid Derivatives as Prodrugs

As described above, the present invention provides the use if alipid-based delivery system for administration of an active drugsubstance selected from lysolipid derivatives, wherein the active drugsubstance is present in the lipid-based system in the form of a prodrug,said prodrug being a lipid derivative having (a) an aliphatic group of alength of at least 7 carbon atoms and an organic radical having at least7 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermorebeing a substrate for extracellular phospholipase A2 to the extent thatthe organic radical can be hydrolytically cleaved off, whereas thealiphatic group remains substantially unaffected, whereby the activedrug substance is liberated in the form of a lysolipid derivative whichis not a substrate for lysophospholipase for the treatment and/orprevention of parasitic infections in mammals.

Typical parasitic infections in mammals are those where the liver,spleen and bone marrow is targeted.

By the term “active drug substance” is meant any chemical entity whichwill provide a prophylactic or therapeutic effect in the body of amammal, in particular a human.

The term “prodrug” should be understood in the normal sense, namely as adrug which is masked or protected with the purpose of being converted(typically by cleavage, but also by in vivo chemical conversion) to theintended drug substance. The person skilled in the art will recognisethe scope of the term “prodrug”.

The active drug substance is selected from lysolipid derivatives, and asit will be understood from the present description with claims, thelysolipid derivatives will have a therapeutic effect—at least—inconnection with parasitic infections where a local area of the body ofthe mammal has a level of extracellular PLA₂ activity caused by saidparasitic infection which can liberate the lysolipid derivative.

As will be understood from the present description with claims, thelipid derivative will often constitute the prodrug referred to above andthe lysolipid derivative will thereby constitute the active drugsubstance often a monoether lysolipid derivative. It should however beunderstood that this does not exclude the possibility of including otherdrug substances, referred to as second drug substances, in thelipid-based delivery systems, neither does it exclude that the organicradical which can be hydrolytically cleaved by the action ofextracellular PLA₂ can have a certain pharmaceutical effect (e.g. as anauxiliary drug substance or an efficiency modifier as describedelsewhere herein) or act as a label. Furthermore, the pharmaceuticaleffect of the “active drug substance”, i.e. the lysolipid derivative,need not the be the most predominant when a second drug substance isincluded, actually the effect of the second drug substance might verywell be the most predominant as will become apparent in the other mainembodiment (see “Lipid derivative liposomes as drug delivery systems”,below).

The active drug substance (lipolipid derivative) release from theprodrug (lipid derivative) is believed to take place as illustrated inthe following example:

Furthermore, the substituent R² may constitute an auxiliary drugsubstance or an efficiency modifier for the active drug substance andwill simultaneously be released under the action of extracellular PLA₂:

It has been described above under the definition of R² how the group R²can have various independent or synergistic effects in association withthe active drug substance, e.g. as an auxiliary drug substance or anefficiency modifier, e.g. permeability or cell lysis modifier. It shouldbe borne in mind that the groups corresponding to R² (e.g. R²—OH orR²—COOH) might have a pharmaceutical effect which is predominant inrelation the effect of the lysolipid derivative (active drug substance).

Lipid Derivatives Formulated as Liposomes and Micelles

The term “lipid-based drug delivery system” should encompassmacromolecular structures which as the main constituent include lipid orlipid derivatives. Suitable examples hereof are liposomes and micelles.It is presently believed that liposomes offer the broadest scope ofapplications and those have been described most detailed in thefollowing. Although liposomes currently are believed to be the preferredlipid-based system, micellular systems are also believed to offerinteresting embodiments within the present invention.

When used herein, the term “label” is intended to mean a species whichis capable of being administered to the mammalian body and beingdetected by extracorporal means for imaging living tissue. The label maybe selected from radiolabels such as radioisotopes andradioisotope-labeled compounds; radiopaque compounds; fluorescentcompounds, etc. More specific labels are ¹¹¹In, ^(99m)Tc ⁶⁷Ga, ¹¹C; Gd,Mn, iron oxide, argon, nitrogen, Iodine, bromine and barium.

Suitable labels for gamma-scintigraphy are diagnostic radionuclides,such as ¹¹¹In, ^(99m) Tc, ⁶⁷Ga. For practical purposes theradionucleotides are complexed with e.g. chelators such as diethylenetriamine pentaacetic acid (DTPA), hexamethylpropyleneamine oxime(HMPAO), diisopropyl iminodiacetic acid (DISIDA), or even proteins suchas human serum albumin (HSA). Alternatively DTPA or similar chelatingcompounds may be derivatized by the incorporation of a hydrophobicgroup, which can anchor the chelating moiety on the liposome surfaceduring or after liposome preparation.

Suitable labels for X-ray are verografin, ioxaglate, iohexol, iopromide,iomeprol, iopamidol, iopentol, iodixanol, ioversol different nonioniccontrast media, etc. which may be incorporated in liposomes and usedboth for planar X-ray imaging of the liver and spleen and for CTimaging.

Suitable labels for magnetic resonance (MR) imaging are paramagneticions, such as Gd and Mn, and iron oxide coupled to various carriermolecules. E. g. gadolinium diethylenetriamine pentaacetic acid(Gd-DTPA) complex has been demonstrated to be effective contrast agentsfor MR imaging of liver, spleen, and hepatic metastases.

Suitable labels for computed tomography (CT) imaging are iodine,bromine, barium, etc.

Other examples of suitable labels are often given by the selected methodof imaging or detection, examples of which are described in detail inHandbook of Medical Imaging, Vol. 1, 2 and 3, SPIE Press, WashingtonUSA, 2000, eds. Beutel, Konden and van Metter.

The label can be adapted so as to be detectable and optionallyquantifiable by a detection method selected from the group consisting ofpositron emission tomography (PET), X-ray, gamma-scintigraphy, magneticresonance (MR) imaging, computed tomography (CT) imaging andultrasonography.

Thus, the present invention also relates to an image enhancing systems(liposomes or micelles) for use in the present invention relies on lipidderivative having (a) an aliphatic group of a length of at least 7carbon atoms and an organic radical having at least 7 carbon atoms, and(b) a hydrophilic moiety, said lipid-conjugated contrast agentsfurthermore being a substrate for extracellular phospholipase A2 to theextent that the organic radical can be hydrolytically cleaved off,whereas the aliphatic group remains substantially unaffected, wherebythe lipid-conjugated contrast agents is liberated in the form of alysolipid derivative which is not a substrate for lysophospholipase.

In one important variant which advantageously can be combined with theembodiments described herein, the lipid derivative (e.g. the prodrug) isincluded in liposomes either as the only constituent or—which is morecommon—in combination with other constituents (other lipids, sterols,etc.). Thus, the lipid-based systems described herein are preferably inthe form of liposomes, wherein the liposomes are build up of layerscomprising the lipid derivative (e.g. a prodrug).

“Liposomes” are known as self-assembling structures comprising one ormore lipid bilayers, each of which surrounds an aqueous compartment andcomprises two opposing monolayers of amphipathic lipid molecules.Amphipathic lipids (herein i.a. lipid derivatives) comprise a polar(hydrophilic) headgroup region (corresponding to the substituent R³ inthe lipid derivatives) covalently linked to one or two non-polar(hydrophobic) aliphatic groups (corresponding to R¹ and R² in the lipidderivatives). Energetically unfavourable contacts between thehydrophobic groups and the aqueous medium are generally believed toinduce lipid molecules to rearrange such that the polar headgroups areoriented towards the aqueous medium while the hydrophobic groupsreorient towards the interior of the bilayer. An energetically stablestructure is formed in which the hydrophobic groups are effectivelyshielded from coming into contact with the aqueous medium.

Liposomes can have a single lipid bilayer (unilamellar liposomes,“ULVs”), or multiple lipid bilayers (multilamellar liposomes, “MLVs”),and can be made by a variety of methods (for a review, see, for example,Deamer and Uster, Liposomes, Marcel Dekker, N.Y., 1983, 27-52). Thesemethods include Bangham's methods for making multilamellar liposomes(MLVs); Lenk's, Fountain's and Cullis' methods for making MLVs withsubstantially equal interlamellar solute distribution (see, e.g., U.S.Pat. No. 4,522,803, U.S. Pat. No. 4,588,578, U.S. Pat. No. 5,030,453,U.S. Pat. No. 5,169,637 and U.S. Pat. No. 4,975,282); andPapahadjopoulos et al.'s reverse-phase evaporation method (U.S. Pat. No.4,235,871) for preparing oligolamellar liposomes. ULVs can be producedfrom MLVs by such methods as sonication (see Papahadjopoulos et al.,Biochem. Biophys. Acta, 135, 624 (1968)) or extrusion (U.S. Pat. No.5,008,050 and U.S. Pat. No. 5,059,421). The liposome can be produced bythe methods of any of these disclosures, the contents of which areincorporated herein by reference.

Various methodologies, such as sonication, homogenisation, French Pressapplication and milling can be used to prepare liposomes of a smallersize from larger liposomes. Extrusion (see U.S. Pat. No. 5,008,050) canbe used to size reduce liposomes, that is to produce liposomes having apredetermined mean size by forcing the liposomes, under pressure,through filter pores of a defined, selected size. Tangential flowfiltration (see WO 89/08846), can also be used to regularise the size ofliposomes, that is, to produce liposomes having a population ofliposomes having less size heterogeneity, and a more homogeneous,defined size distribution. The contents of these documents areincorporated herein by reference. Liposome sizes can also be determinedby a number of techniques, such as quasi-electric light scattering, andwith equipment, e.g., Nicomp® particle sizers, well within thepossession of ordinarily skilled artisans.

It is quite interesting to note that the lipid derivatives canconstitute the major part of a lipid-based system even if this system isa liposome system. This fact resides in the structural (but notfunctional) similarity between the lipid derivatives and lipids. Thus,it is believed that the lipid derivatives can be the sole constituent ofliposomes, i.e. up to 100 mol % of the total dehydrated liposomes can beconstituted by the lipid derivatives. This is in contrast to the knownmono-ether lysolipides, which can only constitute a minor part of theliposomes.

Typically, as will be described in detail below, liposomesadvantageously include other constituents which may or may not have apharmaceutical effect, but which will render the liposome structure morestable (or alternatively more unstable) or will protect the liposomesagainst clearance and will thereby increase the circulation time therebyimproving the overall efficiency of a pharmaceutical including theliposome. The liposomes may also include co-factors such as calcium,which upon release would increase PLA₂ activity, as well as lyso-lipids,fatty acids which are known to increase PLA₂ activity.

This being said, it is believed that the particular lipid derivativeswill typically constitute from 15-100 mol %, such as 50-100 mol %,preferably from 75-100 mol %, in particular 90-100 mol %, based on thetotal dehydrated liposome.

The liposomes can be unilamellar or multilamellar. Some preferredliposomes are unilamellar and have diameters of less than about 200 nm,more preferably, from greater than about 50 nm to less than about 200nm.

The liposomes are typically—as known in the art—prepared by a methodcomprising the steps of: (a) dissolving the lipid derivative in anorganic solvent; (b) removing the organic solvent from the lipidderivative solution of step (a); and (c) hydrating the product of step(b) with an aqueous solvent so as to form liposomes.

The method may further comprise a step of adding an second substance(see below) to the organic solvent of step (a) or the aqueous phase ofstep (c).

Subsequently, the method may comprise a step of extruding the liposomesproduced in step (c) through a filter to produce liposomes of a certainsize, e.g. 100 nm.

The liposomes comprising lipid derivatives may (in principle)exclusively consist of the lipid derivatives. However, in order tomodify the liposomes, “other lipids” may be included as well. Otherlipids are selected for their ability to adapt compatible packingconformations with the lipid derivative components of the bilayer suchthat the all the lipid constituents are tightly packed, and release ofthe lipid derivatives from the bilayer is inhibited. Lipid-based factorscontributing to compatible packing conformations are well known toordinarily skilled artisans and include, without limitation, acyl chainlength and degree of unsaturation, as well as the headgroup size andcharge. Accordingly, suitable other lipids, including variousphosphatidylethanolamines (“PE's”) such as egg phosphatidylethanolamine(“EPE”) or dioleoyl phosphatidylethanolamine (“DOPE”), can be selectedby ordinarily skilled artisans without undue experimentation. Lipids maybe modified in various way, e.g. by headgroup derivatisation withdicarboxylic acids, such as glutaric, sebacic, succinic and tartaricacids, preferably the dicarboxylic acid is glutaric acid (“GA”).Accordingly, suitable headgroup-derivatised lipids includephosphatidylethanolamine-dicarboxylic acids such as dipalmitoylphosphatidylethanolamine-glutaric acid (“DPPE-GA”), palmitoyloleoylphosphatidylethanolamine-glutaric acid (“POPE-GA”) and dioleoylphosphatidylethanolamine-glutaric acid (“DOPE-GA”). Most preferably, thederivatised lipid is DOPE-GA.

The total content of “other lipids” will typically be in the range of0-30 mol %, in particular 1-10 mol %, based on the total dehydratedliposome.

Sterolic compound included in the liposome may generally affects thefluidity of lipid bilayers. Accordingly, sterol interactions withsurrounding hydrocarbon groups generally inhibit emigration of thesegroups from the bilayer. An examples of a sterolic compound (sterol) tobe included in the liposome is cholesterol, but a variety of othersterolic compounds are possible. It is generally believed that thecontent of sterolic compound, if present, will be in the range of 0-25mol %, in particular 0-10 mol %, such as 0-5 mol %, based on the totaldehydrated liposome.

Although the an element of idea on which the present invention residesis that the liposomes or micelles should be taken up by the RES system,it may be advantageous to include a small fraction of lipids or lipidderivatives on which polymeric chains are attached in order to adjustthe rate at which the liposomes and micelles are taken up and therebydegraded. Thus, is may be advantageous to partially, but not fully,employ the so-called STEALTH® liposomes (Liposome Technology Inc.,.Menlo Park, Calif.) which include polyethyleneglycol (PEG)-graftedlipids at about 5 mol % of the total dehydrated liposome, or otherlipopolymers carrying hydrophilic polymer chains. The presence of suchpolymers on the exterior liposome surface should slightly delay, but notprevent, the uptake of liposomes by the organs of the RES. If present,the lipopolymers typically constitute 0.1-10 mol % of the totaldehydrated system.

Hydrophilic polymers suitable for use in lipopolymers are those whichare readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. Preferred polymers are thosehaving a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, and more preferably from about 300 daltons toabout 5,000 daltons. In a particularly preferred embodiment, the polymeris polyethyleneglycol having a molecular weight of from about 100 toabout 5,000 daltons, and more preferably having a molecular weight offrom about 300 to about 5,000 daltons. In a particularly preferredembodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)).Polymers may also be defined by the number of monomers therein; apreferred embodiment of the present invention utilises polymers of atleast about three monomers, such PEG polymers consisting of threemonomers (approximately 150 daltons). Other hydrophilic polymers whichmay be suitable for use in the present invention includepolyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline,polyhydroxypropyl methacrylamide, polymethacrylamide,polydimethylacrylamide, and derivatised celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

Glycolipids are lipids to which a hydrophilic polysaccharide chain iscovalently attached. It will be appreciated that glycolipids can beutilised like lipopolymers although the lipopolymers currently presentsthe most promising results.

It is generally believed that the content of lipopolymer, i present,advantageously will be in the range of 0.1-5 mol %, such as 0.2-4 mol %,in particular 0.5-3 mol %, based on the total dehydrated liposome.

Still other ingredients may constitute 0-2 mol %, in particular 0-1 mol%, based on the total dehydrated liposome.

According to an embodiment, the lipid bilayer of a liposome containslipids derivatised with polyethylene glycol (PEG), such that the PEGchains extend from the inner surface of the lipid bilayer into theinterior space encapsulated by the liposome, and extend from theexterior of the lipid bilayer into the surrounding environment (see e.g.U.S. Pat. No. 5,882,679).

The liposome can be dehydrated, stored and then reconstituted such thata substantial portion of its internal contents is retained. Liposomaldehydration generally requires use of a hydrophilic drying protectantsuch as a disaccharide sugar at both the inside and outside surfaces ofthe liposome bilayers (see U.S. Pat. No. 4,880,635). This hydrophiliccompound is generally believed to prevent the rearrangement of thelipids in the liposome, so that the size and contents are maintainedduring the drying procedure and through subsequent rehydration.Appropriate qualities for such drying protectants are that they arestrong hydrogen bond acceptors, and possess stereochemical features thatpreserve the intramolecular spacing of the liposome bilayer components.Alternatively, the drying protectant can be omitted if the liposomepreparation is not frozen prior to dehydration, and sufficient waterremains in the preparation subsequent to dehydration.

Lipid Derivative Liposomes as Drug or Label Carrier Systems

As mentioned above, the liposomes including the lipid derivatives mayalso include second substances, where such second substances may bedrugs or labels. In a particular embodiment, the lipid-based deliverysystem described above is in the form of liposomes wherein a secondsubstance is incorporated. It should be understood that second drugsubstances may comprise pharmaceutically active ingredients which mayhave an individual or synergistic pharmaceutical effect in combinationwith the lipid derivative and lysolipid derivatives. Furthermore, thatwhen the second substance is a label, said label may comprise contrastsubstances or other substances detectable by means of MR, CT,Gamma-scintigraphy or ultrasonography. The term “second” does notnecessarily imply that the pharmaceutical effect of the second drugsubstance is inferior in relation to that of, e.g., the active drugsubstance derived from the prodrug, but is merely used to differentiatebetween the two groups of substances.

This being said, the present invention also provides the use of adelivery system which is in the form of liposomes, and wherein a secondsubstance is incorporated, either as a drug or as a label.

A possible “second drug substance” is any compound or composition ofmatter that can be administered to mammals, preferably humans. Suchagents can have biological activity in mammals. Second drug substanceswhich may be associated with liposomes are well known anti-paraciticcompounds, such as allopurinol, amodiaquine, amphotericin, antifolates,artemether+benflumetol, artemisinin, derivatives, chloroproguanil,Chloroquine, combination of atovaquone and proguanil HCL (salesnameMalarone™), dapsone, Doxycycline, halofantrine, interferon gamma,Licochocone A, Mefloquine, meglumine antimonate, metronidazole,nitrofurantoin derivatives, paromomycin, pentamidine, primaquine,primaquine, Proguanil, (Chloroquine plus proguanil), puromycin,pyrimethamine, pyronaridine, quinine and sodium stibogluconate.

Liposomal second drug substance formulations enhance the therapeuticindex of the second drug substances by reducing the toxicity of thedrug. Liposomes can also reduce the rate at which a second drugsubstance is cleared from the vascular circulation of mammals.Accordingly, liposomal formulation of second drug substance can meanthat less of the drug need be administered to achieve the desiredeffect.

Liposomes can be loaded with one or more second substances bysolubilising the drug or label in the lipid or aqueous phase used toprepare the liposomes. Alternatively, ionisable second substances can beloaded into liposomes by first forming the liposomes, establishing anelectrochemical potential, e.g., by way of a pH gradient, across theoutermost liposomal bilayer, and then adding the ionisable secondsubstance to the aqueous medium external to the liposome (see, e.g.,U.S. Pat. No. 5,077,056 and WO 86/01102).

Methods of preparing lipophilic drug or label derivatives which aresuitable for liposome or micelle formulation are known in the art (seee.g., U.S. Pat. No. 5,534,499 and U.S. Pat. No. 6,118,011 describingcovalent attachment of therapeutic agents to a fatty acid chain of aphospholipid). A micellar formulation of taxol is described inAlkan-Onkyuksel et al., Pharmaceutical Research, 11:206 (1994).

Accordingly, the second drug substance may be any of a wide variety ofknown and possible pharmaceutically active ingredients, but ispreferably a therapeutically and/or prophylactically active substance.Due to the mechanism involved in the degradation of the liposomes, it ispreferred that the second drug substance is one relating to diseasesand/or conditions associated with a localised increase in extracellularPLA₂ activity.

It is envisaged that the second substance will be distributed in theliposomes according to their hydrophilicity, i.e. hydrophilic secondsubstances will tend to be present in the cavity of the liposomes andhydrophobic second substances will tend to be present in the hydrophobicbilayer. Method for incorporation of second substances are know in theart as has been made clear above.

It should be understood from the above, that the lipid derivativesmay—as prodrugs or discrete constituents—posses a pharmaceutical ordiagnostic activity. However, in a particular embodiment, the presentinvention furthermore relates to a lipid based drug or label deliverysystem for administration of an second substance, wherein the secondsubstance is incorporated in the system (e.g. where the second substanceis encapsulated in the interior of the liposome or in the membrane partof the liposome or the core region of micelle), said system includinglipid derivatives which has (a) an aliphatic group of a length of atleast 7 carbon atoms and an organic radical having at least 7 carbonatoms, and (b) a hydrophilic moiety, where the lipid derivativefurthermore is a substrate for extracellular phospholipase A2 to theextent that the organic radical can be hydrolytically cleaved off,whereas the aliphatic group remains substantially unaffected, so as toresult in an organic acid fragment or an organic alcohol fragment and alysolipid fragment, said lysolipid fragment not being a substrate forlysophospholipase.

As above for the system according to the other embodiment, the organicradical which can be hydrolytically cleaved off, may be an auxiliarydrug or label substance or an efficiency modifier for the secondsubstance. It should be understood that the lipid derivative is a lipidderivative as defined further above. Typically, the lipid derivativeconstitutes 15-100 mol %, such as 50-100 mol %, of the total dehydrated(liposome) system.

The present invention relates to the use of any of the lipid-based drugdelivery systems described herein for the preparation of a medicamentfor the treatment of parasitic infections of a mammal, wherein theparasitic infection is characterized by increasing the level of PLA₂ insaid mammal, particularly in the specific tissue of infection,preferably of the liver or spleen.

Furthermore, the present invention relates to the use of any of thelipid-based delivery systems described herein for the preparation of adiagnostic agent for the detection or quantification of parasiticinfections of a mammal, wherein the parasitic infection is characterizedby increasing the level of PLA₂ in said mammal, particularly in thespecific tissue of infection, preferably of the liver or spleen.

Pharmaceutical Preparations and Therapeutic Uses

“Pharmaceutically acceptable carriers” as used herein are those mediagenerally acceptable for use in connection with the administration oflipids and liposomes, including liposomal drug formulations, to mammals,including humans. Pharmaceutically acceptable carriers are generallyformulated according to a number of factors well within the purview ofthe ordinarily skilled artisan to determine and account for, includingwithout limitation: the particular active drug substance and/or seconddrug or label substance used, the liposome preparation, itsconcentration, stability and intended bioavailability; the disease,disorder or condition being treated with the liposomal composition; thesubject, its age, size and general condition; and the composition'sintended route of administration, e.g., nasal, oral, ophthalmic,subcutaneous, intramammary, intraperitoneal, intravenous, orintramuscular. Typical pharmaceutically acceptable carriers used inparenteral drug administration include, for example, D5W, an aqueoussolution containing 5% weight by volume of dextrose, and physiologicalsaline. Pharmaceutically acceptable carriers can contain additionalingredients, for example those which enhance the stability of the activeingredients included, such as preservatives and anti-oxidants.

The liposome or lipid derivative is typically formulated in a dispersionmedium, e.g. a pharmaceutically acceptable aqueous medium.

An amount of the composition comprising an anti-parasitic effectiveamount of the lipid derivative, typically from about 0.1 to about 1000mg of the lipid derivative per kg of the mammal's body, is administered,preferably intravenously. For the purposes of this invention,“anti-parasitic effective amounts” of liposomal lipid derivatives areamounts effective to inhibit, ameliorate, lessen or preventestablishment, proliferation, growth, etc. of parasites in mammals towhich the lipid derivatives have been administered. Anti-parasiticeffective amounts are generally chosen in accordance with a number offactors, e.g., the age, size and general condition of the subject, theparasitic infection being treated and the intended route ofadministration, and determined by a variety of means, for example, doseranging trials, well known to, and readily practiced by, ordinarilyskilled artisans given the teachings of this invention. Anti-parasiticeffective amounts of the liposomal drugs/prodrugs of this invention areabout the same as such amounts of free, nonliposomal, drugs/prodrugs,e.g., from about 0.1 mg of the lipid derivative per kg of body weight ofthe mammal being treated to about 1000 mg per kg. The pharmaceuticalcomposition is preferably administered parenterally by injection,infusion or implantation (intravenous, intramuscular, intraarticular,subcutaneous or the like) in dosage forms, formulations or e.g. suitabledelivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants.

The formulation and preparation of such compositions is well-known tothose skilled in the art of pharmaceutical formulation. Specificformulations can be found in the textbook entitled “Remington'sPharmaceutical Sciences”.

Thus, the pharmaceutical compositions may comprise the active substancesin the form of a sterile injection. To prepare such a composition, thesuitable active substances are dispersed in a parenterally acceptableliquid vehicle which conveniently may comprise suspending, solubilising,stabilising, pH-adjusting agents and/or dispersing agents. Amongacceptable vehicles that may be employed are water, water adjusted to asuitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solutionand isotonic sodium chloride solution.

The aqueous formulation may also contain one or more preservatives, forexample, methyl, ethyl or n-propyl p-hydroxybenzoate.

Toxicity

Toxicity of the liposomes comprising the lipid derivatives can beassessed by determining the therapeutic window “Tw”, which is anumerical value derived from the relationship between the compound'sinduction of hemolysis and its ability to inhibit thegrowth/proliferation of parasites. TW values are defined as HI₅/GI₅₀(wherein “HI₅” equals the concentration of compound inducing thehemolysis of 5% of the red blood cells in a culture, and wherein “GI₅₀”equals the dose of compound inducing fifty percent growth inhibition ina population of parasitic cells exposed to the agent). The higher anagent's HI₅ value, the less hemolytic is the agent—higher HI₅ 's meanthat greater concentrations of compound are required to be present inorder for the compound to induce 5% hemolysis. Hence, the higher itsHI₅, the more therapeutically beneficial is a compound, because more ofit can be given before inducing the same amount of hemolysis as an agentwith a lower HI₅. By contrast, lower GI₅₀'s indicate better therapeuticagents—a lower GI₅₀ value indicates that a lesser concentration of anagent is required for 50% growth inhibition. Accordingly, the higher isits HI₅ value and the lower is its GI₅₀ value, the better are acompound's agent's therapeutic properties.

Generally, when a drug's TW is less than 1, it cannot be usedeffectively as a therapeutic agent. That is, the agent's HI₅ value issufficiently low, and its GI₅₀ value sufficiently high, that it isgenerally not possible to administer enough of the agent to achieve asufficient level of parasite growth inhibition without also attaining anunacceptable level of hemolysis. As the lipid derivative liposomes takeadvantage of the lower extracellular PLA₂ activity in the bloodstreamcompared to the activity in the diseased tissue, it is believed that theTW will be much higher that for normal monoether lysolipids. As thevariance in activity is in orders of magnitude and as the liposomes willbe “trapped” in tissue with a high extracellular PLA₂ activity, it isgenerally believed the TW of the liposomes of the invention will begreater than about 3, more preferably greater than about 5, and stillmore preferably greater than about 8.

The invention will be illustrated by the following non-limitingexamples.

EXAMPLES Example 1

Liposome Preparation

Unilamellar fully hydrated liposomes with a narrow size distributionwere made from 1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine(1-O-DPPC) and di-hexa-decanoyl-sn-glycero-3-phosphocholine (DPPC). DPPCwere obtained from Avanti Polar lipids and 1-O-DPPC were synthesised inour laboratory. Briefly, weighed amounts of DPPC or 1-O-DPPC weredissolved in chloroform. The solvent was removed by a gentle stream ofN₂ and the lipid films were dried overnight under low pressure to removetrace amounts of solvent. Multilamellar vesicles were made by dispersingthe dried lipids in a buffer solution containing: 150 mM KCL, 10 mMHEPES (pH=7.5), 1 mM NaN₃, 30 μM CaCl₂ and 10 μM EDTA. The multilamellarvesicles were extruded ten times through two stacked 100 nm pore sizepolycarbonate filters as described by Mayer et al., Biochim. Biophys.Acta, 858, 161-168.

Heat capacity curves were obtained using a N-DSC II differentialscanning calorimeter (Calorimetry Sciences Corp., Provo) of the powercompensating type with a cell volume of 0.34 mL. Before scanning, theliposome suspension was equilibrated for 50 min in the calorimeter atthe starting temperature. A scan rate of +10° C./h was used. The lipidconcentration was 1 mM. The gel-to-fluid transition of the multilamellarliposomes (MLV) is characterised as a sharp first-order transition, asreflected by the narrow peak in the heat capacity curves shown in FIGS.1 a and 1 b (upper curves) for 1-O-DPPC and DPPC. The sharp peakreflects the transitional behaviour of multilamellar liposomes and is incontrast to the broader gel-to-fluid transition observed for unilamellarliposomes (LUV) (Pedersen et al., 1996, Biophys. J. 71, 554-560) asshown in FIGS. 1 a and 1 b (lower curves) for the unilamellar extruded1-O-DPPC and DPPC liposomes.

Example 2

Phospholipase A₂ Reaction Profile and Lag Time Measurements

Purified snake-venom phospholipase A₂ (PLA₂ from Agikistrodon piscivoruspiscivorus) has been isolated according to the procedure of Maraganoreet al., J. Biol. Chem. 259, 13839-13843. This PLA₂ enzyme belongs to theclass of low-molecular weight 14 kD secretory enzymes which displaystructural similarity to human extracellular phospholipase A₂ indicatinga common molecular mechanisms of the phospholipase catalysed hydrolysisat the lipid-membrane interface (Wery et al., Nature 352, 79-82; Høngeret al. Biochemistry 35, 9003-9006; Vermehren et al., Biochimica etBiophysica Acta 1373, 27-36). Unilamellar fully hydrated liposomes witha narrow size distribution were prepared from1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (1-O-DPPC) andfrom 1-O-DPPC with 5 mol %1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350] (1-O-DPPE-PEG350). Assay conditions for the PLA₂ reactiontime profile shown in FIG. 2 and the lag-time and percent hydrolysisreported in Table 1 were: 0.15 mM unilamellar liposomes, 150 nM PLA₂,150 mM KCL, 10 mM HEPES (pH 7.5), 1 mM NaN₃, 30 μM CaCl₂, and 10 μMEDTA. TABLE 1 Lag-time and percent hydrolysed 1-O-DPPC at 41° C. asdetermined by HPLC. The lipid concentration was 0.150 mM in a 10 mMHEPES-buffer (pH = 7.5). Lag-time 1-O-DPPC Composition (sec) (%) 100%1-O-DPPC 583 79 95% 1-O-DPPC/5% 1-O-DPPE-PEG350 128 73

The catalytic reaction was initiated by adding 8.9 μL of a 42 μM PLA₂(150 nM) stock solution to 2.5 ml of the thermostated liposomesuspension (0.150 mM) equilibrated for 800 sec prior to addition ofPLA₂. The characteristic lag-burst behaviour of PLA₂ towards theliposomes is signalled by a sudden increase in the intrinsicfluorescence from PLA₂ at 340 nm after excitation at 285 nm followed bya concomitant decrease in the 90° light scattering from the lipidsuspension (Hønger et al., Biochemistry 35, 9003-9006). Samples for HPLCanalysis of the amount of non-hydrolysed 1-O-DPPC remaining andconsequently the amount of1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-1-O-PPC)generated were taken before adding PLA₂ and 1200 sec after the observedlag-time. 100 μl aliquots were withdrawn from the lipid suspension andrapidly mixed with 1 ml chloroform/methanol/acetic acid (2:4:1) solutionin order to quench the enzymatic reaction. The solution was washed with1 ml of water and 20 μl of the heavy organic phase was used for HPLC.The HPLC chromatograms in FIG. 3 show the amounts of 1-O-DPPC before andafter (t=3000 sec) the addition of PLA₂ (t=800 sec) to the liposomesuspension. HPLC analysis was made using a 5 μm diol column, a mobilephase composed of chloroform/methanol/water (730:230:30, v/v) and anevaporative light scattering detector. The turnover of the PLA₂catalysed lipid hydrolysis of 1-O-DPPC to lyso-1-O-DPPC was measured byHPLC (see Table 1). The intrinsic enzyme fluorescence and 90° lightscattering were measured as a function of time as shown in FIG. 2.

Example 3

Phospholipase A₂ Induced Release of an Incapsulated Water-Soluble ModelDrug

Multilamellar 1-O-DPPC-liposomes were made in the presence offluorescent calcein in a self-quenching concentration of 20 mM byhydrating a film of1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine in a HEPESbuffer solution at pH=7.5 for one hour at 10° C. above the phasetransition temperature. Unilamellar liposomes were formed by extrudingthe multilamellar liposomes ten times through two stacked 100 nmpolycarbonate filters. The unilamellar liposomes were rapidly cooled toa temperature below the transition temperature, and thecalcein-containing 1-O-DPPC liposomes were separated from free calceinusing a chromatographic column packed with Sephadex G-50.

Assay conditions for the PLA₂ induced calcein release were 25 μMunilamellar 1-O-DPPC-liposomes, 25 nM PLA₂, 150 mM KCL, 10 mM HEPES (pH7.5 or 8.0), 1 mM NaN₃, 30 μM CaCl₂, and 10 μM EDTA. PLA₂ was added attime 900 sec to 2.5 ml of the thermostated 1-O-DPPC-liposome suspensionequilibrated for at least 20 min at 37° C. prior to addition of PLA₂.The percentage of calcein released is determined as: %Release=100×(I_(F(t))−I_(B))/(I_(T)−I_(B)), where I_(F(t)) is themeasured fluorescence at time t after addition of the enzyme, I_(B) isthe background fluorescence, and I_(T) is the total fluorescencemeasured after addition of Triton X-100 which leads to complete releaseof calcein by breaking up the 1-O-DPPC-liposomes. PLA₂ induced at totalrelease of 90 percent of the entrapped calcein in the 1-O-DPPC-liposomesas shown in FIG. 4.

Example 4

Phospholipase A₂ Controlled Permeability Increase of a Target ModelMembrane

Multilamellar model membrane target liposomes were made in the presenceof fluorescent calcein in a self-quenching concentration of 20 mM byhydrating a film of 1,2-O-dioctadecyl-sn-glycero-3-phosphatidylcholines(D-O-SPC) in a HEPES buffer solution at pH=7.5 for one hour at 10° C.above the phase transition temperature (T_(m)=55° C.).

Unilamellar liposomes were made by extruding the multilamellar targetliposomes ten times through two stacked 100 nm polycarbonate filters.The unilamellar liposomes were rapidly cooled to a temperature below thetransition temperature, and the calcein-containing liposomes wereseparated from free calcein using a chromatographic column packed withSephadex G-50. The unilamellar carrier liposomes composed of1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine were preparedas described above. Calcein release from the target liposomes isdetermined by measuring the fluorescent intensity at 520 nm afterexcitation at 492 nm.

The concentrations of D-O-SPC and 1-O-DPPC-liposomes were 25 μM. Snakevenom PLA₂ (Agkistrodon piscivorus piscivorus) was added (25 nM) toinitiate the hydrolytic reaction leading to the formation of1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-1-O-DPPC) andfatty acid hydrolysis products. As calcein is released from the D-O-SPCliposomes, due to the incorporation of the non-bilayer forminglyso-1-O-PPC and fatty acid hydrolysis products into the target lipidmembrane, a linear increase in the fluorescence at 520 nm afterexcitation at 492 nm is observed when calcein is diluted into thesurrounding buffer medium as shown in FIG. 5. The percentage of calceinreleased is determined as described above (see Example 3).

Example 5

Hemolysis Assay

Unilamellar fully hydrated liposomes with a narrow size distributionwere prepared from1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (1-O-DPPC), andfrom 1-O-DPPC with 5 mol %1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350] (1-O-DPPE-PEG350). The lipids were hydrated in phosphatebuffered saline (PBS).1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH₃) in PBSwas included in the assay as a reference.

Hemolysis assay was performed as described by Perkins et al., Biochim.et Biophys. Acta 1327, 61-68. Briefly, each sample was serially dilutedwith PBS, and 0.5 ml of each dilute suspension of 1-O-DPPC liposomeswere mixed with 0.5 ml washed human red blood cells (RBC) [4% in PBS(v/v)]. For controls, 0.5 ml of the red blood cell suspension was mixedwith either 0.5 ml buffer solution (negative hemolysis control) or 0.5ml water (positive hemolysis control). Samples and standard were placedin a 37° C. incubator and agitated for 20 hours. Tubes were centrifugedat low speed (2000×G) for 10 minutes to form RBCs pellets. 200 μl of thesupernatant was quantified by absorbance at 550 nm using a Perkin-Elmer320 scanning spectrophotometer. 100 percent hemolysis was defined as themaximum amount of hemolysis obtained from the detergent Triton X-100.The hemolysis profile in FIG. 6 shows a low hemolysis value (below 5percent) for 2 mM 1-O-DPPC-liposomes. FIG. 6 also shows that lowconcentrations of ET-18-OCH₃ induces a significant degree of hemolysis.

Example 6

Enhancement of Phospholipase A2 Activity by Negatively Charged PolymerGrafted 1-O-lipids

Unilamellar fully hydrated liposomes with a narrow size distributionwere prepared from1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (1-O-DPPC) and1-O-DPPC with 5 mol %1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350] (1-O-DPPE-PEG350), as described in example 2. Assayconditions for the PLA₂ lag-time measurements were 0.15 mM unilamellarliposomes, 150 nM PLA₂, 150 mM KCL, 10 mM HEPES (pH 7.5), 1 mM NaN₃, 30μM CaCl₂, and 10 μM EDTA. The catalytic reaction was initiated by adding8.9 μL of a 42 μM PLA₂ stock solution to 2.5 ml of the thermostatedliposomes suspension equilibrated for 800 seconds at 41° C. prior toaddition of PLA₂. The time elapsed before the onset of rapid enzymaticactivity is determined by a sudden increase in the intrinsicfluorescence from PLA₂ at 340 nm after excitation at 285 nm. The resultsshown in FIG. 7 show a significant decrease in the lag time when 5 mol %of the negatively charged 1-O-DPPE-PEG₃₅₀ is incorporated into the1-O-DPPC liposomes.

Example 7

Preparation of Micelles Composed of 1-O-DPPE-PEG350,DSPE-PEG750/DPPE-PEG750.

Micelles were made from1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350] (1-O-DPPE-PEG350),di-octadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-750 (DSPE-PEG750). Briefly, weighed amounts of the polymer lipidwere dissolved in chloroform. The solvent was removed by a gentle streamof N₂. The lipid films were then dried overnight under low pressure toremove trace amounts of solvent. Micelles were made by dispersing thedried polymer lipids in a buffer solution containing: 150 mM KCL, 10 mMHEPES (pH=7.5), 1 mM NaN₃, 30 μM CaCl₂ and 10 μM EDTA.

Example 8

Permeability Increase of a Target Model Membranes Controlled byPhospholipase A₂ Hydrolysis of Micelles

Multilamellar model membrane target liposomes were made in the presenceof fluorescent calcein in a self-quenching concentration of 20 mM byhydrating a film of 1,2-O-dioctadecyl-sn-glycero-3-phosphatidylcholines(D-O-SPC) in a HEPES buffer solution at pH=7.5 for one hour at 10° C.above the phase transition temperature (T_(m)=55° C.). Unilamellarliposomes were made by extruding the multilamellar liposomes ten timesthrough two stacked 100 nm polycarbonate filters. The unilamellarliposomes were rapidly cooled to a temperature below the transitiontemperature, and the calcein-containing liposomes were separated fromfree calcein using a chromatographic column packed with Sephadex G-50.Micelles composed of1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350] (1-O-DPPE-PEG350),di-octadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-750 (DSPE-PEG750) were prepared as described in example 7.Calcein release from the target liposome is determined by measuring thefluorescent intensity at 520 nm after excitation at 492 nm.

The concentrations of D-O-SPC and polymer lipid micelles were 25 μM.Snake venom PLA₂ (Agkistrodon piscivorus piscivorus) was added (25 nM)to initiate the hydrolytic reaction leading to instant formation of thelysolipid and fatty acid hydrolysis products. As calcein is releasedfrom the D-O-SPC liposomes, due to the incorporation of the non-bilayerforming polymer-lyso-1-O-lipid and fatty acid into the target lipidmembrane, a linear increase in the fluorescence at 520 nm afterexcitation at 492 nm is observed when calcein is diluted into thesurrounding buffer medium as shown in FIG. 8. The percentage of calceinreleased is determined as described in example 3. PLA₂ catalysedhydrolysis of 1-O-DPPE-PEG350 induced the fastest release rate.

Example 9

Hydrolysis of Micelles Composed of DSPE-PEG750

The hydrolysis of micelles composed DSPE-PEG750 was followed by analysisof the amount of stearic acid generated. The catalytic reaction wasinitiated by adding 8.9 μL of a 42 μM PLA₂ (150 nM) stock solution to2.5 ml a thermostated lipid suspension of DSPE-PEG750 (0.150 mM)equilibrated at 45° C. for 600 seconds prior to addition of PLA₂. Thecharacteristic burst behaviour of PLA₂ towards the micelles is signalledby a sudden increase in the intrinsic fluorescence from PLA₂ at 340 nmafter excitation at 285 nm followed by a concomitant decrease in the 90°light scattering from the lipid suspension (Hønger et al., Biochemistry35, 9003-9006). Samples for HPLC analysis of the amount of stearic acidgenerated were taken before adding PLA₂ and 100 sec after the observedlag-time. The HPLC chromatograms in FIG. 9 shows the amount of stearicacid generated 100 sec after the observed lag time (10 sec) at 45° C.The amount (0.156 mM) of stearic acid generated by hydrolysis was equalto 100% hydrolysis of the DSPE-PEG750 polymer-lipids. HPLC analysis wasmade using a 5 μm diol column, a mobile phase composed ofchloroform/methanol/water (730:230:30, v/v) and an evaporative lightscattering detector (see example 2).

Example 10

Model Examples

Liposomes composed of neutral and/or negatively charged phospholipidscan act as versatile drug or label delivery systems targeting parasiticdiseases in tissues with elevated levels of PLA₂ due to the parasiticinfection, organs of special interest are the liver, spleen and bonemarrow. When administered intravenously these liposomes have a strongtendency to accumulate in the liver and spleen due to a combination ofphysicochemical factors relating to the lipid composition of theliposomes and physiological factors involving the rich blood supply andabundance of macrophages in the liver and spleen. In the examples hereinare described an experimental model system illustrating a new principlefor improved drug or label delivery which takes advantage of an elevatedactivity of extracelluar phospholipase A₂ in the infected liver orspleen tissue. The phospholipase A₂ hydrolyses a lipid-based proenhancerin the carrier liposome, producing lyso-phospholipid and free fattyacid, which are shown in a synergistic way to lead to enhanced liposomedestabilisation and drug release at the same time as the permeability ofthe target membrane is enhanced. The proposed system can be madethermosensitive and offers a rational way for developing smartliposome-based delivery systems by incorporating into the carrierspecific lipid-based proenhancers, prodestabilisers or prodrugs thatautomatically become activated by phospholipase A₂ only at the diseasedand infected target sites.

Liposomes are self-assembled lipid systems and their stability istherefore to a large extent controlled by non-specific physicalinteractions. Insight into the molecular control of the physicalproperties of liposomes is therefore important for manipulating andtailoring the liposomal properties in relation to specific drug-deliverypurposes. As an example, the thermally induced gel-fluid lipid phasetransition has been exploited and optimised design systems for enhancedrelease of drugs due to hyperthermia. It would be desirable if anintelligent and versatile drug-delivery system could be designed whichhas built in a dual virtual trigger mechanism of simultaneous (i)enhanced drug release selectively in the infected target tissue and (ii)enhanced transport of the drug or label into the infected cells. Thisprinciple is illustrated schematically in FIG. 10.

By the examples herein is described the development of a simple andoperative experimental biophysical model system which sustains such adual mechanism to be triggered at the infected target sites such asliver and spleen. The model assumes elevated activity of extracellularphospholipase A₂ in the infected tissue as is the case in inflamed andcancerous tissue where the level of extracellular PLA₂ can be manifoldmagnified. Upon exposure to extracellular PLA₂, the phospholipids ofnegatively charged liposomes have been shown to suffer enhancedhydrolysis compared to neutral liposomes. This leads to destabilisationof the liposome and enhanced release of the encapsulated drug or label.The hydrolysis products, lyso-phospholipids and free fatty acids, act inturn as absorption enhancers for drug or label permeation across thetarget membrane. In this way the phospholipids of the carrier liposomebehave as prodestabilisers at the site of the carrier and asproenhancers at the site of the target membrane. Molecular details ofthis principle are illustrated schematically in FIG. 11.

The experimental model system consists of a negatively charged liposomecarrier and a model target membrane. The carrier is a 100 nm unilamellarliposome made of dipalmitoyl phosphatidylcholine lipids (DPPC) with asmall amount (2.5 mol %) of negatively charged lipid of the typedipalmitoyl phosphatidylethanolamine (DPPE)-PEG₂₀₀₀. The target membraneis another liposome made of1,2-O-dioctadecyl-sn-glycero-phosphatidylcholine (D-O-SPC) which is aphospholipid where the acyl linkages of the stearoyl chains are etherbonds. In contrast to DPPC, D-O-SPC is inert towards PLA₂-catalysedhydrolysis thereby mimicking the stability of an intact target cellmembrane toward degradation by its own enzymes. This experimental assay,which permits simultaneous as well as separate investigation of theeffect of destabilisers at the carrier liposomes and the effect ofenhancers at the target membrane, involves entrapment of a water-solublefluorescent calcein model drug in a self-quenching concentration, in theinterior of the non-hydrolysable target liposome, rather than in thecarrier liposome. The enhanced level of extracellular PLA₂ at the targetmembrane can then be simulated by adding extracellular PLA₂ to initiatethe hydrolytic reaction in a suspension of the carrier and targetliposomes. The permeation of calcein across the D-O-SPC target membraneis subsequently monitored by the increase in fluorescence. In order toinvestigate the effect of the presence of small amounts of negativelycharged PEG-lipids in the carrier liposome, a similar experiment wasperformed with conventional bare DPPC liposomes. Furthermore, in orderto compare and discriminate the permeability enhancing effect oflyso-phospholipids from that of free fatty acids, experiments withoutenzymes were carried out where lyso-phospholipids and free fatty acidswere added simultaneously or separately to the target liposomes.

In FIG. 12.a are shown the results for the release of calcein as afunction of time after adding PLA₂ to the system. The reactiontime-course of the particular PLA₂ used has a characteristic lag-burstbehaviour with a so-called lag time which conveniently can be used as ameasure of the enzymatic activity. A dramatic decrease in the lag timeand a concomitant enhancement of the rate of release are observed whenthe carrier liposomes contain the negatively charged, DPPE-PEG₂₀₀₀, inaccordance with previous findings of enhanced extracellular PLA₂degradation of negatively charged polymer-coated liposomes.

These results suggest that the products of the PLA₂-catalysed hydrolysisof the DPPC lipids of the DPPC-liposomal carrier, lyso-phospholipid andfree fatty acid, which are produced in a 1:1 mixture, are incorporatedinto the target membrane, leading to a large increase in membranepermeability. These products, which have very low water solubility, areknown, due to their non-cylindrical molecular shapes, to induce acurvature stress field in the membrane or small-scale lateral phaseseparation which induce membrane defects and increased permeability.This is substantiated by the data in FIG. 13 which show that theaddition of lyso-phospholipid or fatty acid separately to the presenttarget system, in the absence of PLA₂, leads to an increased rate ofcalcein release across the target membrane. However, the crucial findingis that if lyso-phospholipid and free fatty acid are addedsimultaneously in a 1:1 mixture, a dramatic enhancement in the rate ofrelease is observed as shown in FIG. 13. This strongly suggests that thetwo enhancers act in a synergistic fashion, thereby highlighting theunique possibility in exploiting PLA₂-catalysed hydrolysis for combineddestabilisation of the carrier liposome and enhancement of drugtransport across the target membrane. The synergistic effect is furtheraugmented by the fact that extracellular PLA₂ is activated by its ownhydrolysis products revealing the degradable phospholipids of thecarrier liposome as a kind of proactivators.

It should be pointed out that the effect in the present drug-deliverymodel system of using lipids as proenhancers and prodestabilisers viaextracellular PLA₂ activity is dynamic and refers to an intrinsic timescale. This time scale is the effective retention time of the carrierliposomes near the target membrane. The more rapidly the enzyme becomesactive, the faster is the drug release and the larger the drugabsorption during the time which the carrier spends near the target.Furthermore, the faster the enzyme works the more readily it becomesavailable for hydrolysis of other drug-carrying liposomes that approachthe diseased target site. Once it has been established thatextracellular PLA₂ activity can be used to control drug release, severalrational ways open up for intelligent improvements of the proposeddrug-delivery system via use of well-known mechanisms of alteringextracellular PLA₂ activity by manipulating the physical properties ofthe lipid bilayer to which the enzyme is known to be sensitive. Hencethe strategy is to modify certain physical properties of the carrierliposomes without significantly changing their vascular circulationtime. We shall illustrate this general principle by demonstrating theeffects of both a physico-chemical factor, the lipid composition of thecarrier, and an environmental (thermodynamic) factor, the localtemperature at the target site.

Short-chain phospholipids, such as didecanoyl phosphatidylcholine(DCPC), activate extracellular PLA₂. The effect on calcein permeationacross the target membranes induced by incorporation of a small amountof DCPC into the carrier PEG-liposomes is also shown in FIG. 12.a. Therelease is very fast due to an almost instantaneous activation of theenzyme. We have furthermore found that extracellular PLA₂ becomesdeactivated (data not shown) when a large amount of cholesterol (≈20 mol%) is incorporated into liposomes. In contrast we find that a smallamount of cholesterol (≈3 mol %) activates extracellular PLA₂.

Temperature is known to have a dramatic and highly non-linear effect onextracellular PLA₂ activation in the region of the gel-fluid phasetransition of saturated phospholipid bilayers. This effect is not causedby changes in the enzyme but by dramatic lateral structural changes inthe lipid bilayer. It is possible to take advantage of this effect inthe present drug-delivery system as suggested by the data in FIG. 12.b.As the temperature approaches the transition temperature at 41° C., therate of calcein release is progressively enhanced as quantified by thetime of 50% calcein release, t_(50%), shown in the insert to FIG. 12.b.It has previously been suggested that hypertermia could be exploited toenhance drug release, and that local heating at predefined infectedareas could be used to locally destabilise drug-carrying liposomes, byexploiting the enhanced leakiness of liposomes at their phasetransition. In the new model drug-delivery system proposed here, thesethermosensitive possibilities are integrated and fully exploited via thethermal sensitivity of extracellular PLA₂ to the physical properties ofthe carrier liposome. In contrast to the case where the thermic effectcan only be achieved by a local temperature increase using externalheating sources at a predetermined infected site of some minimal size,the PLA₂-controlled release will be enhanced everywhere wheretemperature and extracellular PLA₂ concentration are elevated, e.g. ininfected tissue, independent of the size of the diseased region andwithout requiring a preceding localisation of the diseased tissue.

DPPC, DCPC, D-O-SPC, and DPPE-PEG₂₀₀₀ were obtained from Avanti PolarLipids. Purified snake venom PLA₂ (Agkistrodon piscivorus piscivorus)was a generous gift from dr. R. L. Biltonen. This PLA₂ enzyme belongs tothe class of low-molecular weight, 14 kD secretory enzymes which displaystructural similarity to human extracellular phospholipase A₂.Multilamellar target liposomes in the presence of fluorescent calcein ina self-quenching concentration of 20 mM were made by hydrating a film ofD-O-SPC in a HEPES buffer solution at pH=7.5 for one hour at 10° C.above the phase transition temperature T_(m)=55° C. Unilamellarliposomes were made by extruding the multilamellar liposomes ten timesthrough two stacked 100 nm polycarbonate filters. The unilamellarliposomes were rapidly cooled to a temperature below the transitiontemperature, and the calcein-containing liposomes were separated fromfree calcein using a chromatographic column packed with Sephadex G-50.The unilamellar carrier liposomes of DPPC, DCPC and DPPE-PEG₂₀₀₀ wereprepared in a similar fashion T_(m)=41° C.). Calcein release from thetarget liposomes is determined by measuring the fluorescent intensity at520 nm after excitation at 492 nm. All measurements are performed attemperatures where the lipids of both the carrier and target liposomesare in the gel state.

Example 11

Phospholipase A₂ Concentration Dependent Release Assay

Multilamellar 1-O-DPPC-liposomes with 10 mol % 1-O-DPPE-PEG350 were madein the presence of fluorescent calcein in a self-quenching concentrationof 20 mM by hydrating a film of 90%1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholine and 10%1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350] in a HEPES buffer solution at pH=7.5 for one hour at 10° C.above the phase transition temperature. Unilamellar liposomes wereformed by extruding the multilamellar liposomes ten times through twostacked 100 nm polycarbonate filters. The unilamellar liposomes wererapidly cooled to a temperature below the transition temperature, andthe calcein-containing liposomes were separated from free calcein usinga chromatographic column packed with Sephadex G-50.

Assay conditions for the PLA₂ induced calcein release were 25 μMunilamellar liposomes, 50, 1 and 0.02 nM PLA₂, 150 mM KCL, 10 mM HEPES(pH 7.5), 1 mM NaN₃, 30 μM CaCl₂, and 10 μM EDTA. PLA₂ was added to 2.5ml of the thermostated micelle suspension equilibrated for at least 300sec at 35.5° C. prior to addition of PLA₂. The percentage of calceinreleased is determined as: % Release 100×(I_(F(t))−I_(B))/(I_(T)−I_(B)),where I_(F(t)) is the measured fluorescence at time t after addition ofthe enzyme, I_(B) is the background fluorescence, and I_(T) is the totalfluorescence measured after addition of Triton X-100 which leads tocomplete release of calcein by breaking up the 1-O-DPPC-liposomes. FIG.14 show that the induced release of calcein was slowest when only 0.02nM PLA₂ was added to the liposome suspension.

Example 12

Hydrolysis of Negatively Charged Liposomes by Phospholipase A2 inCell-Free Rat Peritoneal Fluid

Cell-free peritoneal fluid from rat with casein-induced acuteinflammation was prepared by injecting 5 ml 1% sodium caseinate into theperitoneal cavity of a SRPD male rat, weighing 250-260 g. The rat wassacrificed by bleeding after 24 hours and the inflammatory fluid wascollected from the peritoneum and centrifuged at 1500 G for 20 min inorder to obtain a cell-free peritoneal fluid.

Negatively charged fully hydrated unilamellar liposomes with a narrowsize distribution were prepared from 89 mol %di-hexadecanoyl-sn-glycero-3-phosphoglycerol (DPPG), 10 mol %1-O-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350] (1-O-DPPE-PEG350) and 1 mol %1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (bis-py-DPC).Bis-py-DPC is a PLA₂ substrate with two adjacent pyrene fluorophoresthat form excited-state dimers (eximers) emitting at 470 nm uponexcitation at 342 nm. Phospholipase catalysed hydrolysis separates thetwo fluorophores, which then emit at 380 nm (monomers).

FIG. 15 shows the emission spectra obtained after excitation at 342 nmof bis-py-DPC incorporated in negatively charged liposomes (0.100 mM)before and after adding 100 nM PLA₂ (Agkistrodon piscivorus piscivorus).The observed changed in the emissions spectrum after phospholipasemediated hydrolysis is used in a continuos assay, measuring the eximeremission at 470 nm simultaneously with the monomer emission at 380, uponexcitation at 342 nm. FIG. 16 shows the reaction time profile of ratphospholipase A₂ catalysed hydrolysis of the negatively chargedliposomes. The catalytic reaction was initiated by adding cell-freeperitoneal fluid to 2.5 ml of a thermostated liposome suspensionequilibrated for 60 sec prior to addition of PLA₂. The characteristiclag-burst behaviour of the phospholipase is signalled by a suddenincrease in the monomer fluorescence at 380 nm and a subsequent decreasein the eximer fluorescence as shown at the insert on FIG. 16.

Assay conditions for the PLA₂ reaction time profile shown in FIG. 16were: 0.100 mM unilamellar negatively charged liposomes, 100 μlundiluted cell-free peritoneal fluid, 10 mM HEPES (pH 7.5), 5 mM CaCl₂,and 150 mM NaCl.

1-35. (canceled)
 36. A method for the treatment of parasitic infectionsof a mammal, preferably a human, said parasitic infection beingcharacterised by increasing the PLA₂ level in said mammal byadministering to the mammal an efficient amount of a lipid-based drugdelivery system comprising an active drug substance selected fromlysolipid derivatives, wherein the active drug substance is present inthe lipid-based system in the form of a prodrug, said prodrug being alipid derivative having (a) an aliphatic group of a length of at least 7carbon atoms and an organic radical having at least 7 carbon atoms, and(b) a hydrophilic moiety, said prodrug furthermore being a substrate forextracellular phospholipase A2 to the extent that the organic radicalcan be hydrolytically cleaved off, whereas the aliphatic group remainssubstantially unaffected, whereby the active drug substance is liberatedin the form of a lysolipid derivative which is not a substrate forlysophospholipase.
 37. A method according to claim 36, wherein theincreased level of PLA₂ is localized to a specific tissue and/or organof the mammal, said tissue and/or organ being infected by the parasite.38. A method according to claim 36, wherein the parasitic infectioninvolves the liver and/or the spleen of the mammal.
 39. A methodaccording to claim 36, wherein the organic radical which can behydrolytically cleaved off, is an auxiliary drug substance or anefficiency modifier for the active drug substance.
 40. A methodaccording to claim 36, wherein the prodrug is a lipid derivative of thefollowing formula:

wherein X and Z independently are selected from O, CH₂, NH, NMe, S,S(O), and S(O)₂; Y is —OC(O)—, Y then being connected to R² via eitherthe oxygen or carbonyl carbon atom; R¹ is an aliphatic group of theformula Y¹Y²; R² is an organic radical having at least 7 carbon atoms;where Y¹ is—(CH₂)_(n1)—(CH═CH)_(n2)—(CH₂)_(n3)—(CH═CH)_(n4)—(CH₂)_(n5)—(CH═CH)_(n6)—(CH₂)_(n7)—(CH═CH)_(n8r)—(CH₂)_(n9),and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 to29; n1 is zero or an integer of from 1 to 29, n3 is zero or an integerof from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero oran integer of from 1 to 14, and n9 is zero or an integer of from 1 to11; and each of n2, n4, n6 and n8 is independently zero or 1; and Y² isCH₃ or CO₂H; where each Y¹—Y² independently may be substituted withhalogen or C₁₋₄-alkyl, R³ is selected from phosphatidic acid (P0₂—OH),derivatives of phosphatidic acid and bioisosters to phosphatic acid andderivatives thereof.
 41. A method according to claim 40, wherein R² isan aliphatic group of a length of at least 7 carbon atoms.
 42. A methodaccording to claim 41, wherein R² is a group of the formula Y¹Y².
 43. Amethod according to claim 36, wherein at least a fraction of the prodrugis of the formula defined in claim 40, wherein R³ is a derivative ofphosphatidic acid to which a polymer selected from polyethylene glycol,poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolicacid) copolymers, polyvinyl alcohol, polyvinylpyrrolidone,polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatised celluloses,is covalently attached.
 44. A method according to claim 36, wherein theprodrug constitutes 15-100 mol % of the total dehydrated lipid-basedsystem.
 45. A method according to claim 36, wherein the lipopolymer, ifpresent, constitutes 0.110 mol % of the total dehydrated system.
 46. Amethod according to claim 36, wherein the lipid-based system is in theform of liposomes.
 47. A method according to claim 36 which is in theform of liposomes wherein a second drug substance is incorporated.
 48. Amethod according to claim 47, wherein the second drug substance is atherapeutically and/or prophylactically active substance selected from(i) anti-parasitic agents, (ii) antibiotics and antifungals, and (iii)antiinflammatory agents.
 49. A method according to claim 36, wherein theparasitic infection is caused by a parasitic organism selected from thegroup consisting of Leishmania, Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale, Entamoeba histolytica and Chlornorchis sinensis.
 50. Amethod according to claim 36 for systemic treatment.
 51. A methodaccording to claim 50 for intravenous administration. 52-66. (canceled)67. A method for detecting or quantifying parasitic infections of amammal, preferably a human, said parasitic infection being characterisedby increasing the PLA₂ level in said mammal, by administering to themammal an efficient amount of a lipid based drug delivery systemcomprising a second substance which is a label, said system includinglipid derivatives which has (a) an aliphatic group of a length of atleast 7 carbon atoms and an organic radical having at least 7 carbonatoms, and (b) a hydrophilic moiety, where the lipid derivativefurthermore is a substrate for extracellular phospholipase A2 to theextent that the organic radical can be hydrolytically cleaved off,whereas the aliphatic group remains substantially unaffected, so as toresult in an organic acid fragment or an organic alcohol fragment and alysolipid fragment, said lysolipid fragment not being a substrate forlysophospholipase and said second substance being a label.
 68. A methodaccording to claim 67, wherein the increased level of PLA₂ is localizedto a specific tissue and/or organ of the mammal, said tissue and/ororgan being infected by the parasite.
 69. A method according to claim67, wherein the parasitic infection involves the liver and/or the spleenof the mammal.
 70. A method according to claim 67, wherein the lipidderivative is a lipid derivative of the following formula:

wherein X and Z independently are selected from O, CH₂, NH, NMe, S,S(O), and S(O)₂; Y is —OC(O)—, Y then being connected to R² via eitherthe oxygen or carbonyl carbon atom; R¹ is an aliphatic group of theformula Y¹Y²; R² is an organic radical having at least 7 carbon atoms;where Y¹ is—(CH₂)_(n1)—(CH═CH)_(n2)—(CH₂)_(n3)(CH═CH)_(n4)—(CH₂)_(n5)—(CH═CH)_(n6)—(CH₂)_(n7)—(CH═CH)_(n8r)—(CH₂)_(n9),and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 to29; n1 is zero or an integer of from 1 to 29, n3 is zero or an integerof from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero oran integer of from 1 to 14, and n9 is zero or an integer of from 1 to11; and each of n2, n4, n6 and n8 is independently zero or 1; and y² isCH₃ or CO₂H; where each Y¹—Y² independently may be substituted withhalogen or C₁₋₄-alkyl, R³ is selected from phosphatidic acid (P0₂—OH),derivatives of phosphatidic acid and bioisosters to phosphatic acid andderivatives thereof.
 71. A method according to claim 70, wherein R² isan aliphatic group of a length of at least 7 carbon atoms.
 72. A methodaccording to claim 70, wherein R² is a group of the formula Y¹Y².
 73. Amethod according to claim 67, wherein at least a fraction of the prodrugis of the formula defined in claim 70, wherein R³ is a derivative ofphosphatidic acid to which a polymer selected from polyethylene glycol,poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolicacid) copolymers, polyvinyl alcohol, polyvinylpyrrolidone,polymethoxaoline, polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatised celluloses,is covalently attached.
 74. A method according to claim 67, wherein thelipid derivative constitutes 15-100 mol % of the total dehydratedsystem.
 75. A method according to claim 67, wherein the lipopolymer, ifpresent, constitutes 0.110 mol % of the total dehydrated system.
 76. Amethod according to claim 67, wherein the system is in the form ofliposomes.
 77. A method according to claim 67, wherein the parasiticinfection is caused by a parasitic organism selected from the groupconsisting of Leishmania major Friedlin, Leishmania (viannia) gr.,Leishmania mexicana, Leishmania tropica, Leishmania donovani (infantum),Leishmania aethiopica, Leishmania amazonensis, Leishmania enriettii,Leishmania chagasi Leishmania pifanoi, Plasmodium falciparum, Plasmodiumvivax, Plasmodium ovale, Plasmodium malariae, Trypanosoma cruzi (causingChargas' disease), Trypanosoma brucei, Trypanosoma equiperdum,Trypanosoma evansi Entamoeba histolytica and Chlomorchis sinensis.
 78. Amethod according to claim 67 for systemic treatment.
 79. A methodaccording to claim 78 for intravenous administration.
 80. A methodaccording to claim 67, wherein the label is selected from the groupconsisting of ¹¹¹In, ^(99m)Tc, ⁶⁷Ga, ¹¹C; Gd, Mn, iron oxide, argon,nitrogen, Iodine, bromine and barium.